Gene interference vector- and iron nanoparticle-based composition for killing cancer cells, and use thereof

ABSTRACT

Disclosed in the present invention are a gene interference vector- and iron nanoparticle-based composition for killing cancer cells, and the use thereof. The composition comprises a gene interference vector and iron nanoparticles, wherein the gene interference vector is a CRISPR/Cas13a expression vector or microRNA expression vector controlled by a cancer cell specific promoter DMP, with the Cas13a-gRNA or microRNA expressed by the vector being able to inhibit, in a targeted manner, intracellular iron metabolism and the expression of reactive oxygen related genes, and the iron nanoparticles can be degraded after entering cells to produce iron ions and to increase the reactive oxygen level. The composition comprising the gene interference vector and the iron nanoparticles of the present invention can be used for preparing a new reagent for treating cancers.

TECHNICAL FIELD

The invention relates to the field of cancer gene therapy biotechnology,in particular to a composition for killing cancer cells based on a geneinterference vector and iron nanoparticles and its application.

BACKGROUND TECHNOLOGY

Cancer is an important disease that troubles human health and threatenshuman life. In the long-term battle against cancer, human beings havedeveloped a variety of cancer treatment methods such as surgery,chemotherapy, targeted therapy, and immunotherapy, and have also madesignificant progress, and the cancer survival rate has beensignificantly improved. However, the current level of cancer treatmentis still far from the ideal for the majority of patients in terms ofhealth and life. Therefore, actively developing new cancer treatmenttechnologies is still the goal of continuous efforts in the medicalfield. Gene therapy is a frontier field and a technological highland offuture medicine. The progress made by gene therapy in the field ofgenetic disease treatment has attracted the attention of the medicalcommunity, but there has been no significant breakthrough in the use ofthis technology in cancer treatment. Therefore, the exploration ofcancer single gene therapy also advances progress and breakthrough inthe development of new cancer treatment technologies.

Cancer gene therapy is to introduce genetic material into cancer cells,and use the genetic material to achieve a therapeutic result, interferewith the growth of cancer cells or kill cancer cells. In particular, theintroduction of a certain gene into cells can inhibit cancer cellgrowth, or causing cancer cells apoptosis and necrosis, by expressinggene products such as interfering RNA or protein. Two critical issues ingene therapy, are 1) the selection of genes, which directly determinesthe efficiency of treatment; and 2) to control the expression of genesonly in cancer cells, that is, cancer cell specificity. Relativelyspeaking, the selection of genes is not very challenging. Based on thecurrent genomic science research on a large number of gene functions,many genes, especially genes encoding interfering RNAs and their tumorsuppressor proteins, will more or less, after being introduced intocells, inhibit the growth on cancer cells. The more critical issue ishow to control the gene expression only in cancer cells, that is, cancercell specificity. At present, based on the principles of syntheticbiology, some gene switches have been developed to control the specificexpression of genes in cancer cells, but these gene control elements arecomplex in composition, low in efficiency, and still far fromapplication.

As a fundamental biological phenomenon of cells, programmed cell death(PCD) plays an important role in eliminating unwanted or abnormal cellsin multicellular organisms, which is essential for normal development,homeostasis, and the prevention of hyperproliferative diseases (such ascancer) is crucial. Recently, as a new type of PCD, ferroptosis hasattracted more and more attention. In 2012, Stockwell et al. identifiedferroptosis as an iron-dependent form of non-apoptotic regulated celldeath. Ferroptosis is dependent on intracellular iron, independent ofother metals, and is morphologically, biochemically and genetically notrelated to other well-known regulated cell death types such asapoptosis, necrosis, necroptosis and autophagy. However, ferroptosis isassociated with elevated levels of intracellular reactive oxygen species(ROS), which can be prevented by iron chelation or genetic inhibition ofcellular iron uptake. Inactivation of cellular components by glutathioneperoxidase 4 (GPX4) induces an iron-dependent form of cell death, asthis leads to the accumulation of ROS on membrane lipids.

Although ROS have been shown to regulate cell survival, high levels ofROS can cause irreversible cellular damage, leading to apoptosis,autophagy, and necrosis in various types of cancer cells. So far, manystudies have demonstrated that certain natural products can generatespecific anti-cancerous effects on breast cancer cancel cells byup-regulating ROS levels, suggesting that ROS may mediate selectiveactivation of apoptosis to specifically kill cancer cells. When ferrousiron (Fe²⁺) exists together with peroxides and oxygen, ROS can begenerated through the Fenton reaction. Iron is not only directlyinvolved in many reactions related to ferroptosis, but is alsoresponsible for the accumulation of ROS mediated by the Fenton reaction,as demonstrated by increased iron uptake and inhibition by ironchelators. ROS levels are usually balanced by the combination ofantioxidant production and iron transport systems, typically includingtransferrin uptake, ferritin storage, and ferroportin (FPN). Threeproteins, including transferrin (Tf), transferrin receptor 1 (TFR1) andFPN, play key roles in regulating the balance of iron content in thebody.

Tumors have a particularly high demand for iron compared to normal cellsdue to the unique physiological processes of cancer cells. Usingopportunistic nutritional acquisition is considered as one of thehallmarks of cancer cells. Numerous studies have shown that cancer cellstend to upregulate TFR1 expression to increase iron uptake anddownregulate FPN expression to reduce iron efflux and increase ironretention. However, under iron overload conditions, cancer cells aremore likely to accumulate ROS than normal cells, thereby exacerbatingferroptosis. Therefore, the regulation of iron may provide newtherapeutic opportunities for cancer. To date, FPN is the only knowncellular exporter of iron in mammals. Recently, FPN has been found to bedysregulated in many cancers, such as breast, prostate, ovarian,colorectal, and multiple myeloma, and leukemia cell lines have also beenassociated with low FPN expression relative to normal bone marrow.

Although iron absorption, storage, and excretion are well regulated,administration of iron in nanoparticle form provides an unnatural routefor iron to enter cells. Many studies have reported that iron-basednanomaterials can accumulate at tumor sites due to their ability topassively and actively target, and that iron released as ferrous (Fe²⁺)or iron (Fe³⁺) ions in acidic lysosomes participates in the Fentonreaction and induces ferroptosis to kill cancer cells. However, due tothe importance of iron to cells, cells have evolved a set of mechanismsand systems to maintain intracellular iron balance (iron homeostasis);under the action of this system, cells can effectively export excessiron ions in cells; therefore although a large number of literatureshave reported that iron-based nanoparticles have the effect of causingferroptosis, due to the role of the cellular iron homeostasis system,the iron ions released by iron-based nanoparticles in cells will soon beexported out of cells, so simply using iron-based nanoparticles havevery limited effect on inhibiting the growth of cancer cells byutilizing the iron apoptosis mechanism, and have no clinical developmentvalue.

SUMMARY OF THE INVENTION

Object of the invention: In view of the existing problems of cancer genetherapy and iron nanomaterials inducing ferroptosis of cancer cells, thepresent invention discloses a composition for killing cancer cells andits application. The present invention specifically discloses34 acomposition of gene interference vector and iron nanoparticles forkilling cancer cells, the present invention also provides a new methodfor killing cancer cells using the same. The new method comprises usinggene interference vector and iron nanoparticle. The particle is acombination of two biological and chemical materials to kill cancercells.

Technical solution: in order to achieve the above purpose, a compositionfor killing cancer cells according to the present invention ischaracterized in that it comprises a gene interference vector and ironnanoparticles, and the gene interference vector is a cancer cellspecific promoter DMP-controlled CRISPR/Cas13a expression vector ormicroRNA expression vector.

Wherein, the Cas13a-gRNA expressed by the CRISPR/Cas13a expressionvector, or the microRNA expressed by the microRNA expression vector cantarget and inhibit the expression of target genes in cells,specifically, can target and inhibit intracellular iron metabolism andreactive oxygen species-related gene expression.

The iron nanoparticles are iron nanomaterials that can be degraded togenerate iron ions after entering cells and lead to an increase in alevel of intracellular reactive oxygen species.

Preferably, the iron nanomaterials are ferric oxide nanoparticles(Fe₃O₄@DMSA) modified by Dimethylaminosulfanilide (DMSA) (FeNPs forshort).

Further, the cancer cell-specific promoter DMP promoter is a NF-κBspecific promoter, which is formed by linking an NF-κB decoy and aminimal promoter (patent application number CN201710812983.2), and theDMP promoter can activate its downstream genes to be expressed invarious cancer cells except in normal cells (patent application numbersCN201711335257.2, CN201810163823.4); the DMP promoter can control theexpression of CRISPR/Cas13a or microRNA expression vector in cancercells specific expression.

Wherein, in the CRISPR/Cas13a expression vector, the expression ofCas13a is controlled by the DMP promoter, and the expression of gRNA iscontrolled by the U6 promoter (Chinese Patent Application No.202010096220.4). The functional DNA elements and sequences of theCRISPR/Cas13a expression vector are shown in FIG. 1 . The expression ofmicroRNA is controlled by the DMP promoter in the microRNA expressionvector (Chinese patent application number 201710812983.2); thefunctional DNA elements and sequence of the microRNA expression vectorare shown in FIG. 2 (usually microRNA can be abbreviated as miRNA).

Preferably, the DNA sequence of the functional element of theCRISPR/Cas13a expression vector (pDMP-Cas13a-U6-gRNA; pDCUg forabbreviation) is shown in SEQ ID NO.1; the microRNA expression vector(pDMP-miR) The DNA sequence of the functional element is shown in SEQ IDNO.2.

Further, the CRISPR/Cas13a or microRNA expression vector can expresseither gRNA or microRNA targeting a single gene, or can co-express gRNAor microRNA targeting multiple genes.

Preferably, the iron metabolism and reactive oxygen species relatedgenes mainly include FPN, LCN2, FSP1, FTH1, GPX4, NRF2 and SLC7A11genes.

Further, the CRISPR/Cas13a or microRNA expression vector can expressgRNA or microRNA targeting FPN, LCN2, FSP1, FTH1, GPX4, NRF2 and SLC7A11genes; the gRNA can form a first complex with Cas13a protein, and themicroRNA can interact with RISC forming a second complex, and both thefirst and second complexes can target cleavage of the mRNA of theabove-mentioned genes, resulting in a decrease in the expression levelof the proteins encoded by the above-mentioned genes.

Preferably, the target binding sequences of the gRNA targeting FPN andLCN2 are:

(FPN) 5′-CACCG CAAAG TGCCA CATCC GATCT CCC-3′ and (LCN2)5′-TAACT CTTAATGTTG CCCAG CGTGA ACT-3′;the target binding sequences of the microRNAs targeting FPN, LCN2, FSP1,FTH1, GPX4, NRF2 and SLC7A11 genes are:

(FPN) 5′-TCTAC CTGCA GCTTA CATGAT-3′, (LCN2)5′-TAATG TTGCC CAGCG TGAAC T-3′, (FSP1) 5′-CAAAC AAACA AATAA AGTGG A-3′,(FSP1) 5′-TAAAC AAACA AACAA ATAAA G-3′, (FTH1)5′-ATCCC AAGAC CTCAA AGACA A-3′, (FTH1) 5′-TAAGG AATCT GGAAG ATAGC C-3′,(GPX4) 5′-TTCAG TAGGC GGCAA AGGCG G-3′, (GPX4)AGGAA CTGTG GAGAG ACGGT G-3′, (NRF2) 5′-TACTG ATTCA ACATA CTGAC A-3′,(NRF2) 5′-TTTAC ACTTA CACAG AAACT A-3′, (SLC7A11)5′-AAATG ATACA GCCTT AACAC A-3′, and (SLC7A11)5′-TTGAG TTGAG GACCA GTTAG T-3′.

Preferably, the iron nanoparticles or iron nanomaterials areDMSA-modified Fe₃O₄ nanoparticles (FeNPs) or PEI-modified Fe₃O₄nanoparticles (FeNCs). The two iron nanomaterials can be prepared orobtained commercially.

Furthermore, under the combined action of gene interference vector andiron nanoparticles, the levels of iron ions and reactive oxygen speciesin cancer cells can be sharply increased, which can induce significantiron apoptosis in cancer cells.

Wherein, the gene interference vector can be administered in vivo in theform of a viral vector or a non-viral vector; the iron nanoparticles canbe administered in vivo as a separate chemical material, or can besimultaneously administered as a nanocarrier of a gene interferencevector in vivo.

Preferably, the viral vector is an adeno-associated virus (AAV), and thenon-viral vector is a nanocarrier.

Further, nanocarriers are iron nanoparticles that can bind DNA.

Preferably, the DNA-binding iron nanoparticles are polyethylenimine(PEI) modified iron tetroxide nanoparticles (Fe₃O₄@PEI) (FeNCs forabbreviation).

The application of the composition for killing cancer cells of thepresent invention in the preparation of novel cancer treatment reagents.Specifically, it refers to the application of the combination of twobiological and chemical materials, gene interference vectors and ironnanoparticles, in the preparation of novel cancer treatment reagents.

Specifically, the reagent includes two components: a gene interferencevector and iron nanoparticles; the gene interference vector includes aDMP-controlled CRISPR/Cas13a or microRNA expression vector; the geneinterference vector can be either plasmid DNA or linear DNA; whereiniron nanoparticles include various iron nanoparticles, preferably, ironnanoparticles are FeNPs and FeNCs; wherein FeNCs have dual functions,being both iron nanoparticles and a nanocarrier for gene interferencevectors.

In the present invention, a composition for killing cancer cells isdeveloped, which includes a gene interference vector and ironnanoparticles and a new method for killing cancer cells based on thegene interference vector and iron nanoparticles. The invention combinesthe iron-based nanomaterials with the gene expression regulationtechnology controlled by the NF-κB specific promoter DMP. Controlled bythe DMP promoter are two gene expression interference tools,CRISPR/Cas13a and microRNA, which inhibit the expression of ironmetabolism and reactive oxygen species (ROS)-related genes in cancercells, and cooperate with iron nanoparticles to degrade after enteringcells to form iron ions and generate free radicals, resulting in a sharpincrease in the levels of intracellular iron ions and ROS, resulting insignificant ferroptosis in cancer cells. In the present invention, theexpression of two iron metabolism-related genes FPN and Lcn2 in threeleukemia cells KG-1a, HL60 and WEHI-3 is firstly inhibited by theDMP-controlled CRISPR/Cas13a and microRNA expression vector, and ironnanoparticles are combined Significantly increased the level of ROS inleukemia cells, causing significant ferroptosis in leukemia cells.Multiple cancer cell lines representing 10 common solid tumors were thentreated in the same way with similar results. Explain that thecomposition of the present invention and the treatment method thereofnot only have an anti-cancerous effect on blood cancer cells, but alsohave a anti-cancerous effect on various solid tumor cancer cells.Therefore, the composition of the present invention and the method forkilling cancer cells are: A new technology for broad-spectrum cancercell killing. In addition, the DMP-controlled CRISPR/Cas13a and microRNAexpression vectors targeting FPN and Lcn2 genes were packaged into AAVviruses, which were combined with the same intravenous injection of ironnanoparticles to significantly inhibit the proliferation of leukemiacells in mice. It shows that the proliferation of cancer cells can beinhibited both in vitro and in vivo. Therefore, the composition based onthe gene interference vector and iron nanoparticles for killing cancercells proposed by the present invention, the combination of the twobiological and chemical materials, the gene interference vector and theiron nanoparticles, has potential application value in the preparationof novel cancer therapeutic agents.

Beneficial effect: compared with the prior arts, the present inventionhas the following advantages.

1. Compared with the existing causing cancer death technology that hasbeen clinically applied, the present invention discloses a newanti-cancer cell composition in principle, that is, a composition forGene Interfered Forroptosis Therapy (GIFT) enhanced by geneinterference, in another words, a composition based on a geneinterference vector and iron nanoparticles for killing cancer cells.

Iron-based nanoparticles have been successfully used as MRI imaging inclinical diagnosis of cancer and clinical treatment of anemia, butiron-based nanoparticles have not been used in clinical treatment ofcancer based on their chemical nature. However, a large number ofstudies have shown that iron-based nanoparticles are degraded in theacidic environment of intracellular lysosomes to release iron ions,which in turn leads to increased intracellular ROS levels and induceapoptosis. This process coincides with the mechanism of ferroptosis thathas been extensively studied and revealed in recent years. However, dueto the importance of iron to cells, cells have evolved a set ofmechanisms and systems to maintain intracellular iron balance (ironhomeostasis); under the balance action of this system, cells caneffectively export excess iron ions out of the cells; therefore althougha large number of literatures have reported that iron-basednanoparticles have the effect of causing ferroptosis, due to the effectof the cellular iron homeostasis system, the iron ions released byiron-based nanoparticles in cells will soon be exported to cells, sosimply using iron-based nanoparticles have very limited effects oninhibiting the growth of cancer cells by utilizing the iron apoptosismechanism, and have little clinical value.

The inventors of the present invention found that when cells weretreated with DMSA-modified Fe₃O₄ nanoparticles (FeNPs), FeNPs wouldenter cells and degrade in the acidic environment of lysosomes,releasing iron ions, resulting in an increase in the level ofintracellular iron ions; in order to maintain iron at a steady state,cells respond to increased iron efflux-related gene expression, mostnotably FPN and Lcn2.

In the present invention, FPN and Lcn2 are used as important targets forkilling cancer cells by the mechanism of iron apoptosis. It is believedthat in the case of knocking down the expression of these two ironexport-related genes, treating cells with iron nanomaterials will causean increase in the level of intracellular iron ions; since the generatediron ions cannot be effectively exported out of the cells, it will causea massive accumulation of internal iron ions and the sharp increase ofROS, thus induce significant ferroptosis in cells, but this mechanismworks for both normal cells and cancer cells. Therefore, the mostcritical question is how to control to suppress (or knock down) theexpression of these two iron export-related genes only in cancer cellswithout disturbing the expression of these two genes in normal cells.

In the applicant's past research, a cancer cell-specific promoter, theDMP promoter, was designed and demonstrated, which was formed by linkingthe NF-κB decoy (Decoy) sequence with the Minimal Promoter (Int. J.Biochem. Cell. Biol. 2018, 95:43-52; Patent Application No.CN201710812983.2), and proved that this promoter can drive itsdownstream expression in various cancer cells, but not in normal cells(Hum Gene Ther. 2019, 30:471-484; Gene Therapy 2020, DOI:https://doi.org/10.1038/s41434-020-0128-x; patent application numbersCN201711335257.2, CN201810163823.4). Therefore, the present inventionuses DMP to control the expression of gene interference tools such asCRISPR/Cas13 and miRNA in cells to specifically knock down theexpression of iron export-related genes FPN and Lcn2 in cancer cellswithout affecting their expression in normal cells.

Based on the above reasoning, in the present invention, a cancer cellkilling composition based on a gene interference vector and ironnanoparticles is proposed. The composition combines iron-basednanomaterials with a gene expression interference tool controlled by acancer cell-specific promoter DMP. In the present invention, the DMPpromoter is used to control the intracellular expression of two geneinterference tools, CRISPR/Cas13 and miRNA, and a gene knockdown vectortargeting FPN and Lcn2 mRNA is constructed. Using these vectors incombination with a type of iron nanoparticles (FeNPs), the effect ofthis combination on various cancer cells as well as normal cells wasobserved. The results showed that this combination had a significantanticancerous effect on various cancer cells, while it had no effect onnormal cells. And it is proved that neither of the two alone can producesignificant anticancerous effect on cancer cells, nor does it havesignificant effect on normal cells. By packaging the gene knockdownvector into AAV virus and injecting the resulting recombinant virus withFeNPs into mice intravenously, it was found that the combination of thetwo can significantly inhibit the growth of subcutaneous xenografts inmice. These results fully demonstrate the feasibility and reliability ofthe compositions and method thereof. In addition, by measuring theintracellular iron content, ROS level, and the expression of theeffector gene Cas13 and two target genes in vitro and in vivo, it wasfurther demonstrated that the mechanism of the composition and methodfor killing cancer cells is the genetic interference enhanced ironForroptosis Therapy (GIFT) designed by the present invention.

2. The novel composition and its novel method for killing cancer cellsproposed by the present invention have three significant advantages,namely cancer cell specificity, significant efficacy and applicable to abroad spectrum of cancer cells.

In the present invention, the expression system of CRISPR/Cas13a andmiRNA controlled by DMP is firstly targeted to inhibit the expression oftwo iron metabolism-related genes FPN and Lcn2 in three leukemia cellsKG-1a, HL60 and WEHI-3. Together with iron nanoparticles, it is foundthe composition significantly increased the levels of ROS in leukemiacells, causing significant ferroptosis in leukemia cells. Multiplecancer cell lines representing 10 common solid tumors (14 in total) weresubsequently treated with the same approach, yielding similar results.These experiments demonstrate that the composition and the method forkilling cancer cells not only have works on blood cancer cells, but alsohave can cause death on various solid tumor cancer cells. Therefore, thecomposition and the method for cancer cell treatment are a novelbroad-spectrum anti-cancer cell technology. Experimental studies haveshown that all kinds of cancer cells are basically killed in 72 hoursafter the composition of the present invention and the treatment areadministered, and the death rate of the cancer cells is extremelysignificant. By treating three normal cells (human normal hepatocytesHL7702, human embryonic fibroblasts MRC5 and human gastric mucosaepithelial cells GES-1), it was demonstrated that the new compositionand its anti-cancer treatment method have no effect on the growth ofnormal cells. This experiment and its significant impact demonstrate thecancer cell specificity of the new composition and its anti-cancertreatment method. In addition, in vivo experiments also showed thatCas13a and microRNA controlled by DMP are only expressed in tumortissues, but not in normal tissues, indicating that the anti-cancer celltreatment including the composition and the method for proposed by thepresent invention also has cancer cell specificity in vivo, that is, itonly works in tumor tissue.

3. The composition and the new anti-cancer treatment method proposed bythe present invention is flexible and feasible in terms ofadministration means and dosage forms for anti-cancer treatment in vivo.

To demonstrate the possibility and feasibility of in vivo administrationfor anti-cancer cells treatment in vivo, the present invention hascarried out three batches of animal experiments, respectively trying todeliver gene interference vector DNA in vivo by using viral vectors andiron nanomaterials as non-viral vectors. In the first batch of animalexperiments, rAAV virus and FeNPs were injected intravenously twice, andFeNPs were injected the next day after rAAV injection; in the secondbatch of animal experiments, in order to further simplify theadministration method, rAAV and FeNPs were injected. After mixing invitro, it is administered by one-time intravenous injection. The resultsshowed that the two sequential administrations of rAAV and FeNPs and theone-time simultaneous administration of the two generate similarantitumor effects, which provides a more convenient pathway for the invivo application of the composition disclosed herein and its method forkilling cancer cells.

Traditionally, DNA delivery systems are divided into viralvector-mediated systems and non-viral vector-mediated systems, wherenon-viral pathways have become powerful and popular research tools forelucidating gene structure, regulation, and function. Virus-mediatedgene delivery systems are currently the main gene delivery systems forin vivo gene therapy due to their high efficiency. For example, severalgene therapies approved by the FDA for clinical treatment, as well as alarge number of clinical studies, all use AAV as a gene delivery tool.However, the most critical drawbacks of virus-mediated gene deliverysystems are the potential immune response and the long cycle and highcost of virus preparation; in addition, since AAV virus is a naturallyoccurring virus in the human body, it is present in many individuals,along with natural antibodies and immune memory against it. The wideapplication of AAV virus in all individuals is very limited.Consequently, a large number of non-viral vector systems have beenresearched and developed over the past decade. These includeMagnetofection™, a magnetic nanotransfection agent for nucleic aciddelivery that has been developed (Ther Deliv. 2011; 2:717-26).

In order to overcome the shortcomings of AAV vectors and make full useof the chemical properties of magnetic nanotransfection materials, thepresent invention also attempts to use iron nanoparticles as plasmid DNAnanocarriers (nanocarriers), called Fe nanocarriers (FeNCs). Cancer cellinhibition assays to further simplify reagent preparation and reducecosts. The FeNCs used in the present invention are Fe₃O₄ nanoparticlesmodified by polyethyleneimine (Polyethylenimine, PEI). In the presentinvention, the magnetic transfection agent can not only be used as acarrier for DNA delivery in vivo, but also can be used as an ironnano-donor. The results of the third batch of animal experiments showedthat FeNCs loaded with plasmid DNA (abbreviated as FeNCs@DNA) could alsosignificantly knock down the expression of FPN and Lcn2 genes in tumortissue in mice by intravenous injection, and significantly inhibit tumorgrowth. Therefore, the present invention also develops a new dosage formfor inhibiting the growth of cancer cells by a new method. The dosageform and its two components (plasmid DNA and FeNCs) can not only becommercially manufactured in vitro on a large scale, but also with ashort production cycle at low cost, these characteristic making it avery promising candidate for the development of novel cancertherapeutics. In addition, the dosage form avoids possible immuneresponses from the use of the virus and is expected to be applicable toall individuals.

4. The gene interference vector proposed by the present invention isvery beneficial to the in vivo application of the composition and theanti-cancer treatment method of the present invention.

In the present invention, DMP is used to control two gene interferencesystems CRISPR/Cas13-gRNA and miRNA to achieve the purpose of inhibitingthe expression of target genes in cancer cells in vitro and in vivo, andthe cooperation of DMP and the two gene interference systems is verybeneficial to in vivo application of the new composition and itsanti-cancer treatment methods and create simultaneous multigeneinterference.

In the present invention, the most commonly used and safestadeno-associated virus (AAV) in gene therapy is used as the carrier forgene interference vector (vector) in vivo delivery, but the disadvantageof AVV is that its DNA packaging capacity is limited, and generallycannot package more than 4 Kb DNA fragments. The DMP promoter and thetwo gene interference systems CRISPR/Cas13-gRNA and miRNA used in thepresent invention are very advantageous in the application of AAV for invivo delivery and multigene co-suppression (or knockdown). For example,the present invention co-expresses FPN and Lcn2, and co-expresses other5 target genes (miFFGNS). Since the DMP promoter is very short (84 bp),and Cas13 can process its own gRNA precursor, when constructing gRNAtargeting multiple genes, only one U6 promoter is needed to direct thetranscription of one precursor RNA, and this precursor RNA can beprocessed by Cas13 to form mature gRNA that can target multiple genes ortargets, respectively, such as pDCUg-hFL or pDCUg-mFL in the presentinvention. Being short, an advantage for this DMP promoter andCas13a-gRNA, is very beneficial to package the Cas13 expression vector(DCUg) sequence that can target multiple genes or targets into an AVVparticle, such as the rAAV-DCUg-hFL or rAAV-pDCUg-mFL. The pDMP-miRNAvector used in the present invention is also very advantageous in makinga vector targeting multiple genes or multiple targets. In the pDMP-miRvector used in the present invention, the DMP promoter is only 84 bp,each miRNA backbone is only 341 bp, the HSV TK poly(A) signal is only 49bp, and a complete DMP-miRNA expression unit is only 474 bp in total,which is very beneficial to tandem combination of DMP-miRNA unitstargeting multiple genes or multiple targets to construct co-expressionpDMP-miRNA vectors targeting multiple genes or multiple targets, such aspDMhFL or pDMmFL. This polygenic co-suppression has importantimplications. The present invention finds that the co-expression of gRNAor miRNA targeting multiple iron metabolism or ROS regulation relatedgenes (such as FPN and Lcn2) has a significant synergistic effect inkilling cancer cells, and can be compatible with FeNPs to produce thegreatest cancer cell anticancerous effect (eg pDCUg-hFL or pDCUg-mFL,pDMhFL/pDMmFL).

5. The new composition and anti-cancer treatment method for killingcancer cells proposed by the present invention are expected to solve thedrug resistance problem of cancer cells.

Chemotherapy is one of the main therapies for cancer treatment atpresent, but chemoresistance is still a huge obstacle to cancer cure.Therefore, there is an urgent need to seek new treatment strategies forpeople who no longer benefit from chemotherapy. In addition, currentlyvery popular targeted therapy and immunotherapy have been troubled bytumor drug resistance. In recent years, many studies have reported thatferroptosis is expected to be an important way to solve tumor drugresistance. However, the conventional ferroptosis process is affected bythe active regulation of iron homeostasis and redox homeostasis bycells, and cannot cause cancer cells to undergo ferroptosis with a levelof cancer therapeutic value. Based on a large number of studies oncancer gene therapy and the biological effects of iron nanomaterials,the present invention applies the principle of gene therapy technologyto ferroptosis, and proposes gene interference-enhanced ferroptosistherapy (GIFT) and a new composition and anti-cancer treatment method.Experiments of the present invention demonstrate that all cancer cellstested are almost completely dead after 72 hours of treatment with thenew method.

In summary, the present invention provides a composition for killingcancer cells by combining two biological and chemical materials, a geneinterference carrier and iron nanoparticles, wherein the geneinterference carrier is CRISPR/Cas13a controlled by a cancercell-specific promoter DMP. Or microRNA expression vector, theCas13a-gRNA or microRNA expressed by this vector can target and inhibitintracellular iron metabolism and the expression of reactive oxygenspecies-related genes, and the iron nanoparticles can be degraded togenerate iron ions and increase the level of reactive oxygen speciesafter entering cells. Under the combined action of the gene interferencecarrier and the iron nanoparticle, the invention can lead to a sharpincrease in the levels of iron ions and reactive oxygen species incancer cells and induce significant iron apoptosis in cancer cells. Theproposed combination of gene interference vector and iron nanoparticlesof the present invention can be used to prepare novel cancer therapeuticagents.

DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 is a schematic diagram of the functional DNA elements andsequences of the CRISPR/Cas13a expression vector, wherein in order tovisualize the functional DNA elements and sequences of the vector, theplasmid in the figure is named pDMP-Cas13a-U6-gRNA, abbreviated aspDCUg; the figure shows that DMP controls Cas13a expression, while theU6 promoter controls gRNA expression; the vector is a backbone vectorfor constructing a CRISPR/Cas13a expression vector targeting specificgenes.

FIG. 2 is a schematic diagram of the functional DNA elements andsequences of the microRNA expression vector, wherein the figure showsthat DMP controls the expression of microRNAs, wherein the vector is abackbone vector and is used to construct a microRNA expression vectortargeting specific genes.

FIG. 3 is a schematic diagram of the principle of gene interferenceferroptosis therapy (GIFT), gene expression vector activated by NF-κBand Fe₃O₄ nanoparticles (FeNPs); NF-κB-activated gene expression vectorconsists of a NF-κB-specific promoter (DMP) and its downstreamexpressions, wherein the NF-κB-specific promoter consists of NF-κB decoy(Decoy) sequence and minimal promoter (Minimal Promoter, MP) sequencecomposition; wherein (A) Schematic diagram of the principle of GIFTbased on CRISPR/Cas13a; U6-p is U6 promoter; gRNA is gRNA codingsequence; Cas13a, Cas13a coding sequence; (B) Schematic diagram of theprinciple of miRNA-based GIFT; (C) Quantitative PCR to detect theexpression of NF-κB in different cell lines. ***, p<0.001.

FIG. 4 is a schematic diagram of the effect of FeNPs on cell viability.(A) Effects of FeNPs on the viability of three leukemia cells; threeleukemia cells were treated with different concentrations of FeNPs; cellviability was detected by CCK-8 assay at various times after treatment;(B) The effect of FeNP on the viability of hepatoma cells and two normalcells (HL7702 and MRC-5); cells were treated with differentconcentrations of FeNPs; cell viability was detected by CCK-8 assay atvarious times after treatment.

FIG. 5 is a schematic diagram of the GIFT inhibition experiment of KG-1acells. Cells were transfected with the various plasmids shown in thefigure, cultured for 24 hours, and then cultured for an additional 72hours in a medium with or without 50 μg/mL FeNPs. Cells were stainedwith acridine orange/ethidium bromide and imaged at various time points.Three leukemia cells were treated with various combinations of pDCUg orpDM vector and FeNPs. pDCUg refers to the plasmid of DMP-Cas13a-U6-gRNA,and pDM refers to the plasmid of DMP-miRNA. The plasmid vectors usedinclude pDCUg-NT (gRNA does not target any transcript), pDCUg-hF (gRNAtargets human FPN), pDCUg-hL (gRNA targets human Lcn2), pDCUg-hFL (gRNAtargets human FPN) and Lcn2), pDCUg-mF (gRNA targets mouse FPN),pDCUg-mL (gRNA targets mouse Lcn2), pDCUg-mFL (gRNA targets mouse FPNand Lcn2), pDMNeg (miRNA does not target any transcription present),pDMhF (miRNA targeting human FPN), pDMhL (miRNA targeting human Lcn2),pDMhFL (miRNA targeting human FPN and Lcn2), pDMmF (miRNA targetingmurine FPN), pDMmL (miRNA targeting murine Lcn2) and pDMmFL (miRNAtargeting murine FPN and Lcn2). Cells were transfected with variousplasmids and cultured for 24 h, followed by 72 h in medium with orwithout 50 μg/mL FeNPs. At each time point of FeNPs treatment, cellswere stained with acridine orange/ethidium bromide and imaged. Thefigure shows only representative cell images of plasmids pDMNeg, pDMhFL,pDMmFL, pDCUg-NT, pDCUg-hFL and pDCUg-mFL treated with FeNPs for 72hours.

FIG. 6 is a schematic diagram of the GIFT inhibition experiment of HL60cells. Cells were transfected with the various plasmids shown in thefigure, cultured for 24 hours, and then cultured for an additional 72hours in medium with or without 50 μg/mL FeNPs. Cells were stained withacridine orange/ethidium bromide and imaged at various time points. Thevector annotations are the same as in FIG. 5 .

FIG. 7 is a schematic diagram of the GIFT inhibition experiment ofWEHI-3 cells. Cells were transfected with the various plasmids shown inthe figure, cultured for 24 hours, and then cultured for an additional72 hours in medium with or without 50 μg/mL FeNPs. Cells were stainedwith acridine orange/ethidium bromide and imaged at various time points.The vector annotations are the same as in FIG. 5 .

FIG. 8 is a schematic diagram of quantitative detection of apoptosis ofthree leukemia cells with GIFT inhibitory effect. Cells were treatedwith various combinations of plasmid vectors and FeNPs. Cells werecollected 72 hours after FeNPs administration, and detected by AnnexinV-FITC apoptosis detection kit and flow cytometry. The graph only showsthe final statistical results. The treatments represented by theindividual bars in each histogram on the left, from left to right,correspond to the various treatments from top to bottom in theannotation graph on the right. Representative flow cytometry images areshown in FIG. 9 . All values are mean±s.e.m. where n=3. *, p<0.05; **,p<0.01; ***, p<0.001.

FIG. 9 is a schematic diagram showing the apoptosis of three leukemiacells treated with GIFT by flow cytometry and this figure shows arepresentative flow cytometry image.

FIG. 10 is a schematic diagram of the GIFT inhibition experiment ofHepG2 cells. Cells were transfected with the various plasmids shown inthe figure, cultured for 24 hours, and then cultured for an additional72 hours in medium with or without 50 μg/mL FeNPs. Cells were stainedwith acridine orange/ethidium bromide and imaged at various time points.Cells were treated with various combinations of pDCUg or pDM vector andFeNPs. Plasmid vectors used included pDCUg-NT, pDCUg-hF, pDCUg-hL,pDCUg-hFL, pDMNeg, pDMhF, pDMhL and pDMhFL. Cells were transfected withvarious plasmids and cultured for 24 hours. Cells were then culturedwith medium with or without 50 μg/mL FeNPs for an additional 72 hours.At various time points after FeNPs treatment, cells were stained withacridine orange/ethidium bromide and imaged.

FIG. 11 is a schematic diagram of the GIFT inhibition experiment ofHL7702 cells; cells were transfected with various plasmids in the figureand cultured for 24 hours; the vector transfection of cells was the sameas in FIG. 10 ; cells were induced with or without TNF-α (10 ng/mL) for1 h; cells were then incubated with medium with or without 50 μg/mLFeNPs for an additional 72 h; and cells were stained with acridineorange/ethidium bromide and imaged at various time points.

FIG. 12 is a schematic diagram of the GIFT inhibition experiment ofMRC-5 cells; cells were transfected with various plasmids in the figureand cultured for 24 hours; the vector transfection of cells was the sameas in FIG. 10 ; cells were induced with or without TNF-α (10 ng/mL) for1 h; cells were then incubated with medium with or without 50 μg/mLFeNPs for an additional 72 h; and cells were stained with acridineorange/ethidium bromide and imaged at various time points.

FIG. 13 is a schematic diagram of the apoptosis of HepG2, HL7702 andMRC-5 cells treated with GIFT by flow cytometry; cells were treated withvarious combinations of plasmid vectors and FeNPs; cells were collected72 h after FeNPs administration and detected by flow cytometry withAnnexin V-FITC Apoptosis Detection Kit; the graph only shows the finalstatistical results; the treatments represented by the individual barsin each histogram on the left, from left to right, correspond to thevarious treatments from top to bottom in the annotation graph on theright; representative flow cytometry images are shown in FIG. 14 ; allvalues are mean±s.e.m. wherein n=3. *, p<0.05; **, p<0.01; ***, p<0.001.

FIG. 14 is a schematic diagram of the apoptosis of HepG2, HL7702 andMRC-5 cells treated with GIFT by flow cytometry; this figure shows arepresentative flow cytometry image.

FIG. 15 is a schematic diagram of the GIFT inhibition experiment ofHEK-293T cells; cells were transfected with the various plasmids shownin the figure and cultured for 24 hours; cells were then cultured for anadditional 72 hours in medium with or without 50 μg/mL FeNPs; and cellswere stained with acridine orange/ethidium bromide and imaged at varioustime points.

FIG. 16 is a schematic diagram of the GIFT inhibition experiment of A549cells; cells were transfected with the various plasmids shown in thefigure and cultured for 24 hours; cells were then cultured for anadditional 72 hours in medium with or without 50 μg/mL FeNPs; and cellswere stained with acridine orange/ethidium bromide and imaged at varioustime points.

FIG. 17 is a schematic diagram of the GIFT inhibition experiment ofHT-29 cells; cells were transfected with the various plasmids shown inthe figure and cultured for 24 hours; cells were then cultured for anadditional 72 hours in medium with or without 50 μg/mL FeNPs; and cellswere stained with acridine orange/ethidium bromide and imaged at varioustime points.

FIG. 18 is a schematic diagram of the GIFT inhibition experiment ofPANC1 cells; cells were transfected with the various plasmids shown inthe figure and cultured for 24 hours; cells were then cultured for anadditional 72 hours in medium with or without 50 μg/mL FeNPs; and cellswere stained with acridine orange/ethidium bromide and imaged at varioustime points.

FIG. 19 is a schematic diagram of the GIFT inhibition experiment ofSKOV3 cells; cells were transfected with the various plasmids shown inthe figure and cultured for 24 hours; cells were then cultured for anadditional 72 hours in medium with or without 50 μg/mL FeNPs; and cellswere stained with acridine orange/ethidium bromide and imaged at varioustime points.

FIG. 20 is a schematic diagram of the GIFT inhibition experiment ofMDA-MB-453 cells; cells were transfected with the various plasmids shownin the figure and cultured for 24 hours; cells were then cultured for anadditional 72 hours in medium with or without 50 μg/mL FeNPs; and cellswere stained with acridine orange/ethidium bromide and imaged at varioustime points.

FIG. 21 is a schematic diagram of the GIFT inhibition experiment ofC-33A cells; cells were transfected with the various plasmids shown inthe figure and cultured for 24 hours; cells were then cultured for anadditional 72 hours in medium with or without 50 μg/mL FeNPs, and cellswere stained with acridine orange/ethidium bromide and imaged at varioustime points.

FIG. 22 is a schematic diagram of the GIFT inhibition experiment ofBGC823 cells; cells were transfected with the various plasmids shown inthe figure and cultured for 24 hours; cells were then cultured for anadditional 72 hours in medium with or without 50 μg/mL FeNPs; and cellswere stained with acridine orange/ethidium bromide and imaged at varioustime points.

FIG. 23 is a schematic diagram of the GIFT inhibition experiment ofSGC7901 cells. Cells were transfected with the various plasmids shown inthe figure and cultured for 24 hours; cells were then cultured for anadditional 72 hours in medium with or without 50 μg/mL FeNPs; and cellswere stained with acridine orange/ethidium bromide and imaged at varioustime points.

FIG. 24 is a schematic diagram of the GIFT inhibition experiment ofMGC-803 cells. Cells were transfected with the various plasmids shown inthe figure and cultured for 24 hours; cells were then cultured for anadditional 72 hours in medium with or without 50 μg/mL FeNPs; and cellswere stained with acridine orange/ethidium bromide and imaged at varioustime points.

FIG. 25 is a schematic diagram of the GIFT inhibition experiment ofKYSE450 cells; cells were transfected with the various plasmids shown inthe figure and cultured for 24 hours; cells were then cultured for anadditional 72 hours in medium with or without 50 μg/mL FeNPs; and cellswere stained with acridine orange/ethidium bromide and imaged at varioustime points.

FIG. 26 is a schematic diagram of the GIFT inhibition experiment ofKYSE510 cells; and cells were transfected with the various plasmidsshown in the figure and cultured for 24 hours; cells were then culturedfor an additional 72 hours in medium with or without 50 μg/mL FeNPs;cells were stained with acridine orange/ethidium bromide and imaged atvarious time points.

FIG. 27 is a schematic diagram of the GIFT inhibition experiment ofB16F10 cells; cells were transfected with the various plasmids shown inthe figure and cultured for 24 hours; cells were then cultured for anadditional 72 hours in medium with or without 50 μg/mL FeNPs; and cellswere stained with acridine orange/ethidium bromide and imaged at varioustime points.

FIG. 28 is a schematic diagram of the GIFT inhibition experiment ofHepa1-6 cells; cells were transfected with the various plasmids shown inthe figure and cultured for 24 hours; cells were then cultured for anadditional 72 hours in medium with or without 50 μg/mL FeNPs; and cellswere stained with acridine orange/ethidium bromide and imaged at varioustime points.

FIG. 29 is a schematic diagram of the knockdown effect ofDMP-Cas13a/U6-gRNA and DMP-miR systems; cells were transfected withvarious vectors and incubated for 24 hours, then incubated with orwithout 50 μg/mL FeNPs; cells were detected 48 hours after FeNPsadministration; (A) qPCR analysis of mRNA expression. (B) Western blotanalysis of protein expression; representative images and quantitativeoptical densities are shown; all values are mean±s.e.m. where n=3. *,p<0.05; **, p<0.01; ***, p<0.001.

FIG. 30 is a schematic diagram showing the correlation between theincrease of ROS generation and iron content and GIFT-induced apoptosis;cells were transfected with various plasmids and cultured for 24 hours,followed by an additional 48 hours with or without 50 μg/mL FeNPs;HL7702 and MRC-5 cells were cultured with or without induction of TNF-α(10 ng/mL) for 1 h before treatment with FeNPs; ROS changes and ironcontent were detected 48 hours after FeNPs administration; (A) Flowcytometric analysis of ROS levels; fluorescence shift and quantifiedfluorescence intensity are shown in the figure; treated cells werestained with DCFH-DA using a reactive oxygen species assay kit; ROSchanges indicated by fluorescence shift were analyzed by flow cytometry;(B) Quantitative detection of iron content in cells under varioustreatments; all values are mean±s.e.m. where n=3. *, p<0.05; **, p<0.01;*** p<0.001.

FIG. 31 is a schematic diagram of the cytometry analysis of ROS levelsin GIFT-treated cells; cells were transfected with the various plasmidsshown in the figure and cultured for 24 hours; HL7702 and MRC-5 cellswere induced with or without TNF-α (10 ng/mL) for 1 hour; then with orwithout 50 μg/mL FeNPs; the cells were cultured in the medium for anadditional 48 hours; cells were harvested and stained with DCFH-DA usinga reactive oxygen species assay kit, and flow cytometry was used toanalyze ROS changes indicated by fluorescence shift.

FIG. 32 is a schematic diagram of in vitro assessment of rAAV. KG-1a,WEHI-3 and HL7702 cells were seeded into 24-well plates (1×105cells/well) and cultured for 12 hours; cells were then transfected withvarious viruses in the figure at a dose of 1×105 vg per cell;transfected cells were cultured for 24 hours and then in mediumcontaining or containing 50 μg/mL FeNPs for an additional 72 hours;cells were stained with acridine orange/ethidium bromide and imaged, andparallel cells were analyzed for cell viability with CCK-8; (A)Representative cell images. (B) Cell viability; and all values aremean±s.e.m. where n=3. *, p<0.05; **, p<0.01; ***, p<0.001.

FIG. 33 is a schematic diagram of the transfection of KG-1a cells withFe nanocarriers (FeNCs) loaded with various plasmids; cells (1×105) wereseeded into 24-well plates overnight before transfection; cells weretreated with FeNCs (0.5 μg) loaded with 500 ng of each plasmid accordingto the manufacturer's instructions; the transfected cells were culturedfor 24 hours, and then the cells were incubated with medium with orwithout 50 μg/mL FeNPs for an additional 72 hours; all cells werestained with acridine orange/ethidium bromide at 24, 48 and 72 hoursafter FeNPs administration and imaged under a fluorescence microscope.

FIG. 34 is a schematic diagram of HepG2 cells transfected with Fenanocarriers (FeNCs) loaded with various plasmids; cells (1×105) wereseeded into 24-well plates overnight before transfection; cells weretreated with FeNCs (0.5 μg) loaded with 500 ng of each plasmid accordingto the manufacturer's instructions; the transfected cells were culturedfor 24 hours, and then the cells were incubated with medium with orwithout 50 μg/mL FeNPs for an additional 72 hours; all cells werestained with acridine orange/ethidium bromide at 24, 48 and 72 hoursafter FeNPs administration and imaged under a fluorescence microscope.

FIG. 35 is a schematic diagram of the transfection of KG-1a cells by twokinds of Fe nanocarriers (FeNCs) loaded with various plasmids; cells(1×105) were seeded into 24-well plates overnight before transfection;cells were treated with 50 g/mL FeNCs (FeNCs-1 and FeNCs-2) loaded withvarious plasmids; all cells were cultured for an additional 72 hours;all cells were stained with acridine orange/ethidium bromide at 24, 48and 72 hours after FeNCs administration and imaged under a fluorescencemicroscope; the pDMFL was mixed with FeNCs-1/FeNCs-2 according to theinstructions to form FeNCs-1/FeNCs-2 carrying plasmid pDMFL (denoted asFeNCs-1@pDMFL/FeNCs-2@pDMFL); FeNCs-1@pDMFL/FeNCs-2@pDMFL were added tocells immediately or added to cells after 24 hours (represented asFeNCs-1@pDMFL24h/FeNCs-2@pDMFL24h); FeNCs-1/FeNCs-2 represent two kindsof FeNCs.

FIG. 36 is a schematic diagram of the in vivo antitumor effect of viralvector-based GIFT; (A) Tumor photos of the first and second batch ofanimal experiments; (B) Changes in tumor volume before and aftertreatment; (C) Abundance of viral DNA in various tissues; (D) Ct valuesof Cas13a mRNA detected by qPCR in various tissues; (E) Relativeexpression (RQ) of FPN mRNA in various tissues; (F) Relative expression(RQ) of Lcn2 mRNA in various tissues; All values are mean±s.e.m. wheren=3. *, p<0.05; **, p<0.01; ***, p<0.001.

FIG. 37 is a schematic diagram of the in vivo antitumor effect ofplasmid-loaded iron nanoparticles-based GIFT; (A) Tumor photos of thethird batch of animal experiments; (B) Changes in tumor volume beforeand after treatment; (C) Abundance of plasmid DNA in various tissues;(D) Ct values of Cas13a mRNA detected by qPCR in various tissues; (E)Relative expression (RQ) of FPN mRNA in various tissues; (F) Relativeexpression (RQ) of Lcn2 mRNA in various tissues; and all values aremean±s.e.m. where n=3. *, p<0.05; **, p<0.01; ***, p<0.001.

FIG. 38 is a schematic diagram of the GIFT inhibition experiment ofKG-1a cells; cells were transfected with the various plasmids shown inthe figure and cultured for 24 hours; cells were then cultured for anadditional 72 hours in medium with or without 50 μg/mL FeNPs; cells werestained with acridine orange/ethidium bromide and imaged at various timepoints; plasmids to treat cells include pDMhFSP1-1 (miFSP1-1),pDMhFSP1-2 (miFSP1-2), pDMhFTH1-1 (mihFTH1-1), pDMhFTH1-2 (miFTH1-2),pDMhGPX4-1 (mi GPX4-1), pDMhGPX4-2 (miGPX4-2), pDMhNRF2-1 (miNRF2-1),pDMhNRF2-2 (miNRF2-2), pDMhSLC7A11-1 (miSLC7A11-1) and pDMhSLC7A11-2(miSLC7A11-2) (each abbreviation of the vector is added in theparentheses).

FIG. 39 is a schematic diagram of the GIFT inhibition experiment ofHepG2 cells; cells were transfected with the various plasmids shown inthe figure and cultured for 24 hours; cells were then cultured for anadditional 72 hours in medium with or without 50 μg/mL FeNPs; cells werestained with acridine orange/ethidium bromide and imaged at various timepoints; and the plasmids used to treat the cells are the same as in FIG.38 .

FIG. 40 is a schematic diagram of the GIFT inhibition experiment ofHL7702 cells; cells were transfected with the various plasmids shown inthe figure and cultured for 24 hours; cells were then cultured for anadditional 72 hours in medium with or without 50 μg/mL FeNPs; cells werestained with acridine orange/ethidium bromide and imaged at various timepoints; and the plasmids used to treat the cells are the same as in FIG.38 .

FIG. 41 is a schematic diagram of the GIFT inhibition experiment ofBGC823 cells; cells were transfected with the various plasmids shown inthe figure and cultured for 24 hours; cells were then cultured for anadditional 72 hours in medium with or without 50 μg/mL FeNPs; cells werestained with acridine orange/ethidium bromide and imaged at various timepoints; and the plasmids used to treat the cells are the same as in FIG.38 .

FIG. 42 is a schematic diagram of the GIFT inhibition experiment ofGES-1 cells; cells were transfected with the various plasmids shown inthe figure and cultured for 24 hours; cells were then cultured for anadditional 72 hours in medium with or without 50 μg/mL FeNPs; cells werestained with acridine orange/ethidium bromide and imaged at various timepoints; the plasmids used to treat the cells are the same as in FIG. 38.

FIG. 43 is a schematic diagram of the in vitro antitumor effect of GIFTtargeting other genes; using pDMP-miR vectors targeting 5 genes (FSP1,FTH1, GPX4, NRF2, and SLC7A11), including pDMhFSP1-1 (miFSP1-1),pDMhFSP1-2 (miFSP1-2), pDMhFTH1-1 (mihFTH1-1), pDMhFTH1-2 (miFTH1-2),pDMhGPX4-1 (mi GPX4-1), pDMhGPX4-2 (miGPX4-2), pDMhNRF2-1 (miNRF2-1),pDMhNRF2-2 (miNRF2-2), pDMhSLC7A11-1 (miSLC7A11-1) and pDMhSLC7A11-2(miSLC7A11-2) (abbreviations for each vector in parentheses),transfected 5 cells, 24 hours later; re-cultured with medium with orwithout 50 μg/mL FeNPs cells for 72 hours. Cell viability was analyzedwith CCK-8 at different time points; and all values are mean±s.e.m.where n=3. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Thethree data columns of each treatment in the figure are the detectiondata of 24 hours, 48 hours, and 72 hours from left to right.

DESCRIPTION OF DETAIL EMBODIMENTS

The present invention will be further described below with reference tothe accompanying drawings and embodiments.

EXAMPLES

Gene-interfering ferroptosis therapy (GIFT) inhibits cancer cell growthin vitro and in vivo.

1. Materials and Methods

1.1 Vector Construction

The decoy minimal promoter (DMP), a chemically synthesizedNF-κB-specific promoter containing an NF-κB response sequence and aminimal promoter sequence, was cloned into pMD19-T simple (TAKARA) togive pMD19-T-DMP; the human codon-optimized Cas13a coding sequence wasamplified from pC013-Twinstrep-SUMO-huLwCas13a (Addgene) by PCR, and theamplified product was cloned into pMD19-T-DMP to obtainpMD19-T-DMP-Cas13a; the U6 promoter sequence and the direct repeatsequence (guide RNA, gRNA) of Cas13a separated by the BbsI restrictionsite were chemically synthesized and cloned into pMD19-T-DMP-Cas13a,respectively; obtain pDMP-Cas13a-U6-gRNA (referred to as pDCUg forshort), the DNA sequence of its functional element is shown in SEQ IDNO.1 and FIG. 1 , this vector is a skeleton vector for constructing anexpression vector pdcug-x targeting different gene (x) transcripts.

gRNAs targeting no transcript (NT), human or murine ferroportin (FPN),and Lipocalin 2 (Lcn2) transcripts were designed by CHOPCHOP(http://chopchop.cbu.uib.no/). Complementary oligonucleotides containinga 28 bp gRNA target-specific region and two flanking BbsI sites werechemically synthesized, annealed into double-stranded oligonucleotides,and then cloned into pDCUg by BbsI digestion and ligase. The ligationreaction (10 μL) consisted of 10 units of BbsI enzyme (NEB), 600 unitsof T4 DNA ligase (NEB), 1×T4 DNA ligase buffer, 1 nM double-strandedoligonucleotide, and 50 ng of pDCUg. The ligation reaction was run on aPCR cycler with the following temperature-controlled program: 10 cyclesof 37° C. for 5 minutes and 16° C. for 10 minutes, 37° C. for 30 minutesand 80° C. for 5 minutes. The resulting plasmids were named pDCUg-NT,pDCUg-hFPN/pDCUg-mFPN, pDCUg-hLcn2/pDCUg-mLcn2, respectively. Due to thedifference between human and mouse gene sequences, vectors targetinghuman FPN (hFPN) and Lcn2 (hLcn2) genes, and mouse FPN (mFPN) and Lcn2(mLcn2) genes were constructed respectively (h, human; m, mouse). Inaddition, a pDCUg vector targeting both FPN and Lcn2 genes wasconstructed, named pDCUg-hFL/pDCUg-mFL. Cas13a gRNAs targeting all genesof interest are listed in Table 1.

TABLE 1 gRNA target sequences of Cas13a Name Sequence (5′→3′) PFSHuman FPN CACCGCAAAGTGCCACATCCGATCTCCC T guide Human Lcn2TAACTCTTAATGTTGCCCAGCGTGAACT C guide Mouse FPNTTATTCCAGTTATTGCTGATGCTCCCAT T guide Mouse Len2TTGGTCGGTGGGGACAGAGAAGATGATG T guide No transcriptTAGATTGCTGTTCTACCAAGTAATCCAT N/A guide

The CMV promoter in the pCMV-miR vector was replaced with the DMPpromoter to construct a universal miRNA expression vector pDMP-miR, theDNA sequence of its functional elements is shown in SEQ ID NO. 2 andFIG. 2 . Among them, pCMV-miR was previously constructed by theinventor's laboratory (Int. J. Biochem. Cell. Biol. 2018, 95:43-52),which contains the CMV promoter and its downstream miR backbonesequence. miRNAs targeting human or murine FPN and Lcn2 were designedusing the BLOCK-iT™ RNAi Designer(https://maidesigner.thermofisher.com/rnaiexpress/) program, and thecorresponding oligonucleotides were synthesized by Sangon Biotech. Thesynthesized oligonucleotides were denatured and reannealed to obtaindouble-stranded oligonucleotides, which were then ligated with thelinear pDMP-miR vector cut with BsmBI to generate miRNA expressionvectors targeting FPN and Lcn2 genes, named pDMP, respectively-miR-hFPN/pDMP-miR-mFPN (pDMhF/pDMmF for short) andpDMP-miR-hLcn2/pDMP-miR-mLcn2 (pDMhL/pDMmL for short) Use the symbol “/”to mean “or”). The detection vector was amplified by PCR and verified byDNA sequencing. In addition, a miRNA expression vector that cansimultaneously express (i.e. co-express) both FPN and Lcn2 wasconstructed and named aspDMP-miR-hFPN-DMP-miR-hLcn2/pDMP-miR-mFPN-DMP-miR-mLcn2 (abbreviated aspDMhFL/pDMmFL). In a similar manner, miR-Neg double-strandedoligonucleotides were synthesized and prepared from the sequence ofplasmid pcDNA™6.2-GW/EmGFP-miR-Neg, and ligated into the pDMP-miR vectorto generate pDMP-miR-Neg (abbreviated as pDMNeg), this vector was usedas a negative control vector. The target sequences and chemicallysynthesized oligonucleotide sequences used to construct pDMIP-miRvectors targeting individual genes are shown in Table 2.

The same method was used to design and construct pDMP-miR vectorstargeting other five genes, namely FSP1, FTH1, GPX4, NRF2 and SLC7A11;and miRNAs targeting two targets were designed for each gene. Theconstructed vectors were named pDMhFSP1-1, pDMhFSP1-2, pDMhFTH1-1,pDMhFTH1-2, pDMhGPX4-1, pDMhGPX4-2, pDMhNRF2-1, pDMhNRF2-2,pDMhSLC7A11-1 and pDMhSLC7A11-2, respectively. The target sequences andchemically synthesized oligonucleotide sequences used to constructpDMP-miR vectors targeting individual genes are shown in Table 2.

TABLE 2 Oligonucleotides used to construct pDMP-miRNA vectors targetingdifferent genes Name Sequence (5′→3′) Human miR-FPN-FTGCTGTCTACCTGCAGCTTACATGATGTTTTGGCCACTGACTGACATCATGTACTGCAGGTAGAHuman miR-FPN-RCCTGTCTACCTGCAGTACATGATGTCAGTCAGTGGCCAAAACATCATGTAAGCTGCAGGTAGACMurine miR-FPN-FTGCTGTATACAGACTCACTGATTTGCGTTTTGGCCACTGACTGACGCAAATCAGAGTCTGTATAMurine miR-FPN-RCCTGTATACAGACTCTGATTTGCGTCAGTCAGTGGCCAAAACGCAAATCAGTGAGTCTGTATACHuman miR-Lcn2-FTGCTGTAATGTTGCCCAGCGTGAACTGTTTTGGCCACTGACTGACAGTTCACGGGGCAACATTAHuman miR-Lcn2-RCCTGTAATGTTGCCCCGTGAACTGTCAGTCAGTGGCCAAAACAGTTCACGCTGGGCAACATTACMurine miR-Lcn2-FTGCTGTCAAGTTCTGAGTTGAGTCCTGTTTTGGCCACTGACTGACAGGACTCATCAGAACTTGAMurine miR-Lcn2-RCCTGTCAAGTTCTGATGAGTCCTGTCAGTCAGTGGCCAAAACAGGACTCAACTCAGAACTTGACmiR-Neg-FTGCTGAAATGTACTGCGCGTGGAGACGTTTTGGCCACTGACTGACGTCTCCACGCAGTACATTTmiR-Neg-RCCTGAAATGTACTGCGTGGAGACGTCAGTCAGTGGCCAAAACGTCTCCACGCGCAGTACATTTCHuman miR-Fsp1-1-FTGCTGCAAACAAACAAATAAAGTGGAGTTTTGGCCACTGACTGACTCCACTTTTTGTTTGTTTGHuman miR-Fsp1-l-RCCTGCAAACAAACAAAAAGTGGAGTCAGTCAGTGGCCAAAACTCCACTTTATTTGTTTGTTTGCHuman miR-Fsp1-2-FTGCTGTAAACAAACAAACAAATAAAGGTTTTGGCCACTGACTGACCTTTATTTTTGTTTGTTTAHuman miR-Fsp1-2-RCCTGTAAACAAACAAAAATAAAGGTCAGTCAGTGGCCAAAACCTTTATTTGTTTGTTTGTTTACHuman miR-Fth1-1-FTGCTGATCCCAAGACCTCAAAGACAAGTTTTGGCCACTGACTGACTTGTCTTTGGTCTTGGGATHuman miR-Fth1-1-RCCTGATCCCAAGACCAAAGACAAGTCAGTCAGTGGCCAAAACTTGTCTTTGAGGTCTTGGGATCHuman miR-Fth1-2-FTGCTGTAAGGAATCTGGAAGATAGCCGTTTTGGCCACTGACTGACGGCTATCTCAGATTCCTTAHuman miR-Fth1-2-RCCTGTAAGGAATCTGAGATAGCCGTCAGTCAGTGGCCAAAACGGCTATCTTCCAGATTCCTTACHuman miR-Gpx4-1-FTGCTGTTCAGTAGGCGGCAAAGGCGGGTTTTGGCCACTGACTGACCCGCCTTTCGCCTACTGAAHuman miR-Gpx4-1-RCCTGTTCAGTAGGCGAAAGGCGGGTCAGTCAGTGGCCAAAACCCGCCTTTGCCGCCTACTGAACHuman miR-Gpx4-2-FTGCTGAGGAACTGTGGAGAGACGGTGGTTTTGGCCACTGACTGACCACCGTCTCCACAGTTCCTHuman miR-Gpx4-2-RCCTGAGGAACTGTGGAGACGGTGGTCAGTCAGTGGCCAAAACCACCGTCTCTCCACAGTTCCTCHuman miR Nrf2-1-FTGCTGTACTGATTCAACATACTGACAGTTTTGGCCACTGACTGACTGTCAGTATTGAATCAGTAHuman miR-Nrf2-1-RCCTGTACTGATTCAATACTGACAGTCAGTCAGTGGCCAAAACTGTCAGTATGTTGAATCAGTACHuman miR-Nrf2-2-FTGCTGTTTACACTTACACAGAAACTAGTTTTGGCCACTGACTGACTAGTTTCTGTAAGTGTAAAHuman miR-Nrf2-2-RCCTGTTTACACTTACAGAAACTAGTCAGTCAGTGGCCAAAACTAGTTTCTGTGTAAGTGTAAACHuman miR-SLC7A11-1-FTGCTGAAATGATACAGCCTTAACACAGTTTTGGCCACTGACTGACTGTGTTAACTGTATCATTTHuman miR-SLC7A11-1-RCCTGAAATGATACAGTTAACACAGTCAGTCAGTGGCCAAAACTGTGTTAAGGCTGTATCATTTCHuman miR-SLC7A11-2-FTGCTGTTGAGTTGAGGACCAGTTAGTGTTTTGGCCACTGACTGACACTAACTGCCTCAACTCAAHuman miR-SLC7A11-2-RCCTGTTGAGTTGAGGCAGTTAGTGTCAGTCAGTGGCCAAAACACTAACTGGTCCTCAACTCAAC

DCUg-NT/hFL/mFL and DMNeg/DMhFL/DMmFL sequences were amplified by PCRfrom pAAV-DCUg-NT/hFL/mFL and pAAV-DMNeg/DMhFL/DMmFL, respectively.Using MluI (upstream) and XbaI (downstream) restriction sites, theDCUg-NT/hFL/mFL and DMNeg/DMhFL/DMmFL sequences were cloned intopAAV-MCS (VPK-410, Stratagene) to construct pAAV-DCUg-NT/hFL/mFL andpAAV-DMNeg/DMhFL/DMmFL vectors, respectively.

1.2. Nanoparticles, Cells and Culture

DMSA-coated Fe₃O₄ magnetic nanoparticles (FeNPs) and polyethylenimine(PEI)-modified ferric oxide nanoparticles (FeNCs) were purchased fromNanjing Dongna Biotechnology Co., Ltd.

Cells used in the present invention include KG-1a (human acute myeloidleukemia cells), HL60 (human amyloid acute leukemia cells), WEHI-3(mouse acute monocytic leukemia cells), HepG2 (human liver cancer cells)cells), A549 (human lung cancer cells), HT-29 (human colon cancercells), C-33A (human cervical cancer cells), SKOV3 (human ovarian cancercells), PANC-1 (human pancreatic cancer cells), MDA-MB-453 (human breastcancer cells), BGC-823/MGC-803/SGC-7901 (human gastric adenocarcinomacells), KYSE450/KYSE510 (human esophageal cancer cells), Hepa1-6 (mouseliver cancer cells), B16F10 (mouse melanoma cells), HEK-293T (humanfetal kidney cells), HL7702 (human normal hepatocytes), MRC5 (humanembryonic fibroblasts) and GES-1 (human normal gastric mucosalepithelial cells). Three leukemia cell lines KG-1a, HL60 and WEHI-3 werecultured in IMEM medium (Gibco). HEK-293T, HepG2, Hepa1-6, C-33A,PANC-1, MDA-MB-453, B16F10, MRC-5, GES-1 cells were cultured with DMEMmedium (Gibco). A549, HT-29, SKOV-3, BGC-823/MGC-803/SGC-7901,KYSE450/KYSE510 and HL7702 cells were cultured in RPMI 1640 medium(Gibco). All three media were supplemented with 10% fetal bovine serum(HyClone), 100 units/mL penicillin (Thermo Fisher Scientific), and 100μg/mL streptomycin (Thermo Fisher Scientific). Cells were incubated at37° C. in a humidified incubator with 5% CO₂.

1.3. Cytotoxicity of FeNPs

Determine the optimal dose of nanoparticles. In vitro cytotoxicity ofFeNPs was performed using CCK-8 assay. KG-1a, HL60, WEHI-3, HepG2,HL7702 and MRC-5 cells were seeded into 96-well plates at a density of5000 cells/well, respectively. Cells were cultured overnight and treatedmultiple times with various concentrations (0 μg/mL, 30 μg/mL, 50 μg/mL,100 μg/mL, 150 μg/mL, 200 μg/mL, 250 μg/mL) of FeNPs. Six groups ofcells were used for each treatment, with four replicates per group. 10μL of Cell Counting Kit-8 (CCK-8) solution (BS350B, Biosharp) was addedto each well at different time points after treatment (0 d, 1 d, 2 d, 3d, 4 d and 5 d) middle. After an additional 1 h incubation at 37° C.,the optical density at 450 nm was measured using a microplate reader(BioTek).

1.4. Treatment of Cells with Gene Regulatory Tools and FeNPs

Cells were transfected with plasmids using Lipofectamine 2000 (ThermoFisher Scientific) following the manufacturer's instructions. Briefly,cells (1×105 cells/well) were seeded into 24-well plates overnightbefore transfection. Cells were then transfected with 500 ng of variousplasmids, including pDCUg-NT, pDCUg-hFPN/pDCUg-mFPN,pDCUg-hLcn2/pDCUg-mLcn2, pDCUg-hFL/pDCUg-mFL, pDMNeg, pDMhF/pDMmF,pDMhL/pDMmFL. Transfected cells were incubated for 24 hours, thenincubated with or without 50 μg/mL FeNPs, and cells were incubated foran additional 72 hours. For HL7702 and MRC5, cells were first incubatedwith or without TNF-α (10 ng/mL) for 1 h before treatment with FeNPs.24h, 48h and 72h after FeNPs administration, all cells were stained withacridine orange/ethidium bromide according to the manufacturer'sinstructions. Cells were imaged under a fluorescence microscope (IX51,Olympus) to observe the number of live and dead cells. To quantifyapoptosis, cells were collected 72 h after administration of FeNPs anddetected using the Annexin V-FITC Apoptosis Detection Kit (BD, USA)according to the manufacturer's instructions. Fluorescence intensity ofcells was quantified with a CytoFLEX LX flow cytometer (Beckman).

1.5. Analysis of Reactive Oxygen Species Production

Cells were treated with FeNPs as described in step 1.4. Briefly, cellswere seeded in 24-well plates (1×105 cells/well) and cultured overnight.Cells were then transfected with 500 ng of various plasmids, includingpDCUg-NT, pDCUg-hFL/pDCUg-mFL, pDMNeg, and pDMhFL/pDMmFL. Transfectedcells were cultured for 24 h, followed by an additional 48 h with orwithout incubation with FeNPs at 50 μg/mL. Treated cells were stainedwith 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) using areactive oxygen species assay kit (Beyotime) according to themanufacturer's instructions. Fluorescence shifts were indicative of ROSchanges when analyzed on a CytoFLEX LX flow cytometer (Beckman).

1.6. Iron Content Determination

Cells were processed as in step 1.5. Intracellular iron was determinedby complete digestion of cells 48 hours after FeNPs administration.Cells were washed with PBS (pH 7.0), collected and counted. Cells werethen pelleted by centrifugation, resuspended in 50 μL of 5M hydrochloricacid, and incubated at 60° C. for 4 hours. Cells were centrifuged againand the supernatant was transferred to a 96-well plate. Add 50 μL offreshly prepared detection reagent (0.08% K2S2O8, 8% KSCN and 3.6% HClin water) to each well and incubate the microplate for 10 min at roomtemperature. Absorbance at 490 nm was measured using a microplate reader(BioTek). Iron content was determined by the absorbance obtained afternormalization with a standard curve generated from a FeCl₃ standardsolution. Iron is reported as the mean iron content per cell, calculatedas the mean divided by the number of cells in each sample. Threereplicate wells were set up for each experiment and repeated at leastsix times.

1.7. Western Blot Analysis

Cells were seeded into 6-well plates (2×105 cells/well) and grownovernight. Cells in each well were transfected with 1000 ng of pDCUg-NT,pDCUg-hFL, pDMNeg, and pDMhFL plasmid DNA, respectively. Forty-eighthours after transfection, whole cell extracts were prepared using aphosphoprotein extraction kit (SA6034-100T, Signalway Antibody, USA)according to the manufacturer's instructions. The protein lysate (20μg/sample) was resolved by SDS-PAGE, and the target protein was detectedby Western blot (WB). The antibodies used to detect the target proteinin WB were: GAPDH rabbit monoclonal antibody (ab181602, Abcam, UK),SLC40A1 rabbit polyclonal antibody (ab58695, Abcam, UK), Lipocalin-2rabbit polyclonal antibody (ab63929, Abcam, UK). The secondary antibodywas IRDye 800CW-labeled goat anti-rabbit IgG (Licor). PVDF blots wereimaged using an Odyssey infrared fluorescence imaging system (Licor) andthe fluorescence intensity was quantified.

1.8. Virus Preparation

HEK293T cells were seeded into 75 cm² flasks (5×106 cells/flask) andcultured overnight. Cells were then transfected using Lipofectamine 2000following the manufacturer's instructions, and the transfected DNA wastwo helper plasmids, pHelper and pAAV-RC (Stratagene), and one pAAVplasmid, including pAAV-DCUg-NT, pAAV-DCUg-hFL/pAAV-DCUg-mFL, pAAV-DMNegand pAAV-DMhFL/pAAV-DMmFL. Cells were cultured for an additional 72hours after transfection. The virus was then collected and purified asdescribed in the literature (Gene Therapy, 2020, DOI:10.1038/s41434-020-0128-x). AAV titers were determined by qPCR usingprimers AAV-F/R (Table 3). The quantified virus was aliquoted and storedat −80° C. until use. The obtained viruses were named rAAV-DCUg-NT,rAAV-DCUg-hFL/rAAV-DCUg-mFL, rAAV-DMNeg and rAAV-DMhFL/rAAV-DMmFL.

1.9. Virus Assessment

KG-1a, WEHI-3 and HL7702 cells were seeded into 24-well plates (1×105cells/well) and cultured for 12 hours. Cells were then transfected withrAAV-DCUg-NT, rAAV-DCUg-hFL/rAAV-DCUg-mFL, rAAV-DMNeg, andrAAV-DMhFL/rAAV-DMmFL, respectively, at a virus dose of 1×105 vg percell. Transfected cells were cultured for 24 hours and then incubatedwith or without 50 μg/mL FeNPs for an additional 72 hours. All cellswere stained and imaged with acridine orange/ethidium bromide. Cellviability was detected using the CCK-8 assay (BS350B, Biosharp).

1.10. Iron Nanocarriers (FeNCs)-Based GIFT Inhibits Cancer Cells

In order to verify whether PEI-modified Fe₃O₄ iron nanoparticles (FeNCs)can be used as a delivery vehicle for gene interference carriers tointroduce gene interference carriers into cells, and play the role ofGIFT in inhibiting cancer cells together with iron nanoparticles as genedelivery vehicles, two studies were conducted. cell experiments. Inorder to distinguish iron nanoparticles as gene delivery vehicles fromcommon iron nanoparticles (FeNPs) used above, iron nanoparticles as genedelivery vehicles are defined as iron nanocarriers (FeNCs). Two batchesof FeNCs, named FeNCs-1 and FeNCs-2, were used in the experiments.

In the first FeNCs-based GIFT inhibition of cancer cells, variousplasmids (including pDCUg-NT, pDCUg-hFL, pDMNeg, and pDMhFL) were mixedwith FeNCs-1 (1 μg DNA/μg FeNCs-1), to prepare FeNCs loaded with plasmidDNA, namely FeNCs-1@pDCUg-NT, FeNCs-1@pDCUg-hFL, FeNCs-1@pDMNeg andFeNCs-1@pDMhFL. Cells were seeded into 24-well plates (1×105 cells/well)and cultured overnight. Cells were then treated with FeNCs with orwithout 0.5 μg plasmid DNA loaded and plasmid DNA alone for 24 hours;cells were then cultured with or without 50 μg/mL FeNPs for 72 hours.Differentially treated cells were stained with acridine orange/ethidiumbromide and imaged at different time points (24 hours, 48 hours and 72hours).

In the second FeNCs-based GIFT inhibition of cancer cells, two plasmids(pDMNeg and pDMhFL) were mixed with FeNCs-1 and FeNCs-2 (1 μg DNA/μgFeNCs-1) according to the manufacturer's instructions to prepare theload FeNCs of plasmid DNA, namely FeNCs-1@pDMhFL and FeNCs-2@pDMhFL. Theprepared FeNCs-1@pDMhFL and FeNCs-2@pDMhFL were used to treat cellsimmediately or left at room temperature for 24 hours before treatment.Cells were seeded into 24-well plates (1×105 cells/well) and culturedovernight. Cells were then incubated with or without 50 μg/mL FeNCs(FeNCs alone or FeNCs loaded with plasmid DNA) for an additional 72hours. Differentially treated cells were stained with acridineorange/ethidium bromide and imaged at different time points (24 hours,48 hours and 72 hours).

1.11. Animal Studies

Four-week-old BALB/c female mice with an average body weight of 20 gwere purchased from Changzhou Cavens Laboratory Animal Co. Ltd., China.All animal experiments in the present invention followed the guidelinesand ethics of the Animal Care and Use Committee of Southeast University(Nanjing, China). The tumor-bearing mouse model was established bysubcutaneously transplanting 1×107 WEHI-3 cells into the inner thigh ofBALB/c female mice; after feeding for 1 week, the tumor size wasmeasured in vivo with a precision caliper. Tumor volume was calculatedusing the formula V=(ab2)/2, wherein a is the longest tumor diameter andb is the shortest tumor diameter. Considering that the number of animalsand cells required would be too large to manage if all experimentalgroups were performed at the same time, animal experiments wereperformed in three batches.

In the first batch of animal experiments, tumor-bearing mice wererandomly divided into six treatment groups (PBS, n=6; FeNPs, n=6;rAAV-DCUg-NT, n=6; rAAV-DCUg-NT+FeNPs, n=6; rAAV-DCUg-mFL, n=6;rAAV-DCUg-mFL+FeNPs, n=7) (n is the number of mice). Each group of micewas injected intravenously with PBS (pH7.0), rAAV-DCUg-NT, rAAV-DCUg-NT,rAAV-DCUg-mFL, rAAV-DCUg-mFL, respectively. All viruses were injected ata dose of 1×1010 vg/mouse. On the second day, three of the groups(FeNPs, rAAV-DCUg-NT+FeNPs and rAAV-DCUg-mFL+FeNPs) were intravenouslyinjected with FeNPs at a dose of 3 mg/kg body weight. On day 7 afterFeNPs injection, mice were euthanized and photographed, then tumors wereexcised, and tumor sizes were measured and calculated as describedabove. Mice were dissected and various tissues (including heart, liver,spleen, lung, kidney, and tumor tissues) were collected andcryopreserved in liquid nitrogen.

In the second batch of animal experiments, tumor-bearing mice wererandomly divided into five treatment groups (FeNPs, n=6; rAAV-DMNeg,n=6; rAAV-DMmFL, n=7; rAAV-DMNeg+FeNPs, n=7; rAAV-DMmFL+FeNPs, n=6).Then each group of mice was injected intravenously with FeNPs,rAAV-DMNeg, rAAV-DMmFL, rAAV-DMNeg+FeNPs, rAAV-DMmFL+FeNPs,respectively. The injection doses of all viruses and FeNPs were the sameas the first batch of animal experiments, but in this batch of animalexperiments, rAAV (1×1010 vg/mouse) was first mixed with FeNPs (3 mg/kgbody weight), and then injected intravenously at one time mice. On day 7post-injection, mice were euthanized and photographed, then tumors weredissected, and tumor sizes were measured and calculated as describedabove. The mice were dissected, and various tissues were collected andcryopreserved in liquid nitrogen.

In the third batch of animal experiments, tumor-bearing mice wererandomly divided into six treatment groups (PBS, n=6; FeNCs, n=6;pAAV-DMNeg+FeNCs, n=6; pAAV-DMmFL+FeNCs, n=7; pAAV-DCUg-NT+FeNCs, n=6;pAAV-DCUg-mFL+FeNCs, n=7). Then each group of mice was injectedintravenously with PBS (pH7.0), FeNCs, pAAV-DMNe+FeNCs,pAAV-DMmFL+FeNCs, pAAV-DCUg-NT+FeNCs, pAAV-DCUg-mFL+FeNCs, respectively.The doses of different plasmids and FeNCs were 2 mg/kg body weight and 3mg/kg body weight, respectively. On day 7 after FeNCs injection, micewere euthanized and photographed, then tumors were dissected and tumorsizes were measured and calculated as described above. Mice weredissected and various tissues were collected and cryopreserved in liquidnitrogen.

1.12. Quantitative PCR

Total RNA was isolated from cells or mouse tissues after 48 h incubationwith FeNPs using TRIzol™ (Invitrogen) according to the manufacturer'sinstructions. cDNA was prepared using the FastKing RT kit (TIANGEN)according to the manufacturer's instructions. Genomic DNA (gDNA) wasextracted from various tissues of mice using the TIANamp Genomic DNA Kit(TIANGEN). Target genes were amplified from cDNA and gDNA by qPCR usingHieff qPCR SYBR Green Master Mix (Yeasen). Triplicate samples for eachtreatment were evaluated on an ABI Step One Plus (Applied Biosystems).Relative mRNA transcript levels were compared to the GADPH internalreference and calculated as relative quantity (RQ) according to thefollowing equation: RQ=2−ΔΔCt. Viral DNA abundance was normalized toGADPH internal reference and calculated according to the followingformula: RQ=2−ΔCt. Cas13a mRNA expression levels are shown as Ct values.All experiments were performed in triplicate and repeated at least threetimes.

The expression of NF-κB RelA/p65 in cells was detected by quantitativePCR (qPCR) using primers Human/Murine RelA-F/R and Human/MurineGAPDH-F/R. Results were normalized to GAPDH and analyzed by the 2−ΔCtmethod. According to melting curve analysis, all qPCR primers haveamplification specificity, and their sequences are shown in Table 3.

Table 3 qPCR guide Name Sequence (5′→3′) AAV-F TGCATGACCAGGCTCAGCTAAAV-R GACAGGGAAGGGAGCAGTG Cas13a-F GGAAAAGTACCAGTCCGCCA Cas13a-RGAAGTCCAGGAACTTGCCGA GAPDH-F ATTTGGTCGTATTGGGCG GAPDH-RCTCGCTCCTGGAAGATGG Human RelA-F CCTGGAGCAGGCTATCAGTC Human Re A-RATGGGATGAGAAAGGACAGG Mouse RelA-F TGCGATTCCGCTATAAATGCG Mouse RelA-RACAAGTTCATGTGGATGAGGC Human GAPDH-F ATTTGGTCGTATTGGGCG Human GAPDH-RCTCGCTCCTGGAAGATGG Mouse GAPDH-F TCACCACCATGGAGAAGGC Mouse GAPDH-RGCTAAGCAGTTGGTGGTGCA Human FPN-F CACAACCGCCAGAGAGGATG Human FPN-RCACATCCGATCTCCCCAAGT Human Lcn2-F CCCGCAAAAGATGTATGCCA Human Lcn2-RCTCACCACTCGGACGAGGTA Mouse FPN-F TGGAACTCTATGGAAACAGCCT Mouse FPN-RTGGCATTCTTATCCACCCAGT Mouse Lcn2-F TGGCCCTGAGTGTCATGTG Mouse Lcn2-RCTCTTGTAGCTCATAGATGGTGC

1.13. Statistical Analysis

All data are presented as mean±standard error. Statistical analysis andgraphing were performed using GraphPad Prism software (GraphPadSoftware). Data were statistically processed using analysis of varianceand Student's t-test. Differences at p<0.05 were consideredstatistically significant. *, p<0.05; **, p<0.01; ***, p<0.001.

2. Results

2.1. Principle and process of genetic interference ferroptosis therapy(GIFT) FIGS. 3A and 3B schematically illustrate the principle of geneinterference ferroptosis therapy (GIFT). GIFT consists of a geneexpression regulatory vector activated by transcription factor NF-κB andFe₃O₄ nanoparticles (FeNPs). The NF-κB-activated gene expressionregulatory vector consists of a promoter DMP and downstream effectorgenes, wherein the DMP promoter consists of a NF-κB decoy sequence and aminimal promoter sequence. DMP is a NF-κB specific promoter, and sinceNF-κB is a transcription factor that is overactivated in inflammationand cancer, DMP can be activated by NF-κB in NF-κB-overactivated cancercells, which drives the expression of its downstream effector genes,while in normal cells without NF-κB expression, the DMP promoter cannotbe activated, and its downstream effector genes are not expressed.Therefore, the DMP promoter is a cancer cell-specific activatedpromoter. When the DMP-controlled CRISPR/Cas13a or miRNA gene expressioninterference system is transfected into cancer cells, the overactivatedNF-κB will bind to DMP to drive the expression of Cas13a or miRNA, andthe expressed Cas13a protein can be activated with the U6 promoter. Theexpressed gRNA is assembled into a Cas13a/gRNA complex, and the miRNA isprocessed and bound to the RISC complex. Both Cas13a-gRNA and themiRNA-RISC complex can target and degrade the target mRNA, inhibiting orknocking down the expression of the target gene in cancer cells. In thepresent invention, two genes related to iron metabolism, namely FPN andLcn2, are selected as target genes. The intracellular functions of FPNand Lcn2 are both related to the cellular efflux of iron. Therefore, byreducing the expression of these two genes in cancer cells, the activeefflux of large amounts of iron ions produced by FeNPs can be prevented.The accumulation of iron ions leads to a significant increase in thelevel of intracellular ROS, which in turn leads to significantferroptosis in cancer cells. In normal cells, the expression ofCas13a-gRNA or miRNA, the interference system of the two genes, cannotbe produced, and its expression is not affected. The cells can activelyefflux the iron ions generated after FeNPs enter the cells, and maintainiron homeostasis, thus not affecting normal cells nor making an impact.

2.2. Expression of NF-κB RelA in Cancer Cells and Normal Cells

NF-κB is widely activated in almost all types of tumor cells. Since theactivity of intracellular NF-κB is crucial for the feasibility of thepresent invention, quantitative PCR was first used to detect threeleukemia cells (KG-1a, HL60 and WEHI-3), other 15 cancer cells(including HEK-293T, HepG2, A549, HT-29, C-33A, SKOV3, PANC-1,MDA-MB-453, BGC-823/MGC-803/SGC-7901, KYSE450/KYSE510, Hepa1-6 andB16F10) and two Levels of NF-κB RelA/p65 in human normal cell lines(HL7702 and MRC5). The results showed that different levels of NF-κBRelA/p65 expression were detected in all cancer cell lines, but not innormal cell lines (MRC-5 and HL7702) (FIG. 3C). Therefore, theNF-κB-specific promoter DMP can be used to drive the specific expressionof effector genes in cancer cells.

2.3. In Vitro Effects of FeNPs on Cells

To evaluate the cytotoxic effects of FeNPs, three leukemia cells(including KG-1a, HL60 and WEHI-3), one solid tumor cell HepG2, and twohuman normal cells (HL7702 and MRC5) were incubated with variousconcentrations of FeNPs for five days, and at various time points aftertreatment, cell viability was analyzed using CCK-8, and cell growthcurves were established. The results showed that FeNPs had no obviouscytotoxic effect on all six cell lines when the dose was lower than 50g/mL during the incubation time (FIG. 4 ), but when the dose was greaterthan 50 g/mL, FeNPs had no obvious cytotoxic effect on normal cells. Thegrowth of HL7702 and MRC-5 was significantly affected (FIG. 4B).Furthermore, cancer cells were more tolerant to FeNPs, and FeNPstreatment at 100 μg/mL had no significant effect on both human leukemiacells (KG-1a and HL60) (FIG. 4A), but not on mouse leukemia cells WEHI-3and human hepatoma cells HepG2 have produced significant toxicity (FIG.4B). Therefore, 50 μg/mL was used as a safe dose of FeNPs for furtherstudies, which is equivalent to 3 mg kg-1 intravenously injected inrodents.

2.4. In Vitro Antitumor Effects of Gene-Interfering Ferroptosis Therapy(GIFT)

Firstly, the inhibitory effect of GIFT on leukemia cells wasinvestigated. Use various plasmid vectors including pDCUg-NT,pDCUg-hFPN/pDCUg-mFPN, pDCUg-hLcn2/pDCUg-mLcn2, pDCUg-hFL/pDCUg-mFL,pDMNeg, pDMhF/pDMmF, pDMhL/pDMmL and pDMhFL/pDMmFL vectors, weretransfected with three kinds of leukemia cells (KG-1a, HL60 and WEHI-3)in the 24-well plate respectively. Two-four hours after transfection,the cells were cultured with the culture medium containing live cellsbut not containing 50 μg/mL FeNPs for 24 hours, 48 hours and 72 hoursrespectively, and the cells were detected by acridine orange/ethidiumbromide double staining. Dead and alive, and cells treated in parallelwere collected at the 72-hour time point for quantitative detection ofapoptosis. The results showed that by combining with the vectorspDCUg-hFPN/pDCUg-mFPN, pDCUg-hLcn2/pDCUg-mLcn2, pDCUg-hFL/pDCUg-mFL,pDMhF/pDMmF, pDMhL/pDMmL and pDMhFL/pDMmFL, FeNPs causes three leukemiacells significant apoptosis in all of them (FIG. 5 , FIG. 6 , FIG. 7 );and this anticancerous effect was obviously time-dependent (FIG. 5 ,FIG. 6 , FIG. 7 ). However, each plasmid alone, FeNPs, and thecombination of negative plasmids (pDCUg-NT and pDMNeg) with FeNPs didnot significantly affect all cells at any treatment time (FIG. 5 , FIG.6 , FIG. 7 ). More importantly, FeNPs produced the strongest cancer cellkilling effect when two gene interference vectors (pDCUg-hFL/pDCUg-mFLand pDMhFL/pDMmFL) were co-expressed (FIG. 5 , FIG. 6 , FIG. 7 ), asynergistic effect of the co-interference of the two genes was shown. Inaddition, the quantitative detection of apoptosis of cells with lostcells also showed the same results as the detection of acridineorange/ethidium bromide double staining (FIG. 8 , FIG. 9 ).

Then, the inhibitory effect of GIFT on solid tumor cells was examined.Human hepatoma cells HepG2 were transfected in 24-well plates withvarious plasmid vectors including pDCUg-NT, pDCUg-hFPN, pDCUg-hLcn2,pDCUg-hFL, pDMNeg, pDMhF, pDMhL and pDMhFL vectors, respectively. 24hours after transfection, the cells were cultured with a culture mediumcontaining viable but not containing 50 μg/mL FeNPs for 24 hours, 48hours and 72 hours, respectively, and the cell death was detected byacridine orange/ethidium bromide double staining, and at 72 hours. Cellstreated in parallel were collected at various time points, and apoptosiswas quantified. The results showed that FeNPs caused significantapoptosis of HepG2 cells by combining with the vectors pDCUg-hFPN,pDCUg-hLcn2, pDCUg-hFL, pDMhF, pDMhL and pDMhFL (FIG. 10 ); and thisanticancerous effect also showed obvious time dependence (FIG. 10 ).However, each plasmid alone, FeNPs, and the combination of negativeplasmids (pDCUg-NT and pDMNeg) with FeNPs did not have a significanteffect on HepG2 cells at any treatment time (FIG. 10 ). Similarly, whentwo gene interference vectors (pDCUg-hFL and pDMhFL) were co-expressed,FeNPs produced the strongest cancer cell killing effect (FIG. 10 ), alsoshowing the synergistic effect of the two gene co-interference.

To examine the cancer cell specificity of GIFT, two human normal cells,HL7702 and MRC5, were treated in the same manner as HepG2. The resultsshowed that none of the vectors, alone or in combination with FeNPs, hada significant effect on either (FIG. 11 , FIG. 12 ), which is consistentwith the result that NF-κB expression was not detected in these twonormal cells (FIG. 3C). To further observe the essential role of NF-κBactivation on GIFT, these two cells were firstly transfected withpDCUg-hFL and pDMhFL, and then treated with the NF-κB activator TNF-α,followed by FeNPs. It was found that these two normal cells were alsosignificantly killed by GIFT (FIG. 11 , FIG. 12 ). It shows that onlyNF-κB activation, GIFT can play a role in killing cancer cells. Inaddition, the quantitative measurement of cell apoptosis with the lossof cells also showed the same results as the acridine orange/ethidiumbromide double staining detection (FIG. 13 , FIG. 14 ).

Only NF-κB activation of GIFT was also observed in HEK-293T cells.HEK-293T cells are human embryonic kidney cells transfected with a virusthat expresses the large T antigen. Although these cells are notconsidered to be cancer cells, their NF-κB expression is significantlyactivated (FIG. 3C). Therefore, the combination of pDCUg-hFL and pDMhFLvectors with FeNPs also produced a significant killing effect on thiscell (FIG. 15 ).

To investigate whether the GIFT mechanism has broad-spectrumanti-cancerous effect, a variety of cancer cells representing differenthuman and mouse tumors, including A549, HT-29, C-33A, SKOV3, PANC-1,MDA-MB-453, BGC-823/MGC-803/SGC-7901, KYSE450/KYSE510, Hepa1-6, B16F10,were treated with the same treatment method. Since co-expression of thevectors produced the most significant cancer cell killing effects in thethree leukemia cells and human hepatoma cell HepG2 cell experiments,only the pDCUg-hFL/pDCUg-mFL and pDMhFL/pDMmFL vectors were used inexperiments with more cancer cells. The results showed that when thepDCUg-hFL/pDCUg-mFL and pDMhFL/pDMmFL vectors were combined with FeNPs,they had a significant anti-cancerous effect on various cancer cells(FIG. 16 ˜FIG. 28 ); and this anti-cancerous effect was also criticallytime-dependent (FIG. 16 -FIG. 28 ). Likewise, each vector alone, FeNPs,and the combination of negative plasmids (pDCUg-NT and pDMNeg) withFeNPs did not significantly affect all cells at any treatment time(FIGS. 16-28 ).

To evaluate the knockdown effect of two tools, DMP-Cas13a-U6-gRNA(pDCUg) and DMP-miR (pDMP-miR), the expression levels of FPN and Lcn2genes were detected by qPCR. The results showed that targetinggRNA/miRNA significantly down-regulated the level of target mRNA incancer cells KG-1a, HL60 and HepG2 cells (FIG. 29A). However, no changeswere found in normal cells HL7702 cells, further indicating the NF-κBspecificity of the DMP promoter as well as the cancer cell specificity(i.e., only works in cancer cells). To further explore the specificexpression of effector genes in cancer cells, the expression levels ofCas13a mRNA under various treatments were detected in the above fourcells. The results showed that Cas13a was only expressed in cancer cellsKG-1a, HL60 and HepG2 transfected with pDCUg-hFL and pDMhFL (FIG. 29A);however, Cas13a mRNA was not detected in normal cells HL7702 under alltreatments (FIG. 29A). These results suggest that the DMP-based geneexpression system can be activated in cancer cells but not normal cells,which leads to the specific expression of effector genes only in cancercells. Afterwards, the protein levels of FPN and Lcn2 were detected bywestern blotting (WB). The results showed that the expression of FPN andLcn2 proteins was significantly inhibited in cancer cells KG-1a, HL60and HepG2 transfected with targeting plasmids (pDMhFL and pDCUg-hFL)(FIG. 29B).

Iron-based nanomaterials can upregulate ROS levels through the Fentonreaction, resulting in specific killing effects in cancer. In order toinvestigate whether the Fenton reaction occurs in the co-cultures of thepresent invention and to explore the underlying mechanism ofGIFT-induced apoptosis in cancer cells, we measured the effect of GIFTin three leukemia KG-1a, HL60 and WEHI-3 and one solid tumor cell HepG2in Intracellular active ROS levels and intracellular iron content undertreatment of various plasmids (pDCUg-hFL, pDCUg-NT, pDMhFL and pDMNeg)in combination with FeNPs. The results showed that three leukemia cellsand HepG2 had increased intracellular ROS levels in all treatmentscontaining FeNPs (FIG. 30A; FIG. 31 ), especially when treated withpDCUg-hFL and pDMhFL vectors in combination with FeNPs. ROS levels incancer cells were dramatically elevated (FIG. 30A; FIG. 31 ), which isconsistent with the significant apoptosis of these cancer cells underthe same treatments indicated by the above assays (FIG. 8 ; FIG. 13 ).Intracellular iron content assays showed that the four cancer cells hadincreased intracellular iron content in all treatments containing FeNPs(FIG. 30B), especially when treated with pDCUg-hFL and pDMhFL vectors incombination with FeNPs. The iron content in the cells increaseddramatically (FIG. 30B), which is consistent with the dramatic increasein ROS levels in the four cancer cells under the same treatmentindicated by the above assays. It shows that the inhibition of theexpression of iron efflux-related target genes FPN and Lcn2 in cancercells combined with FeNPs can cause a sharp increase in theintracellular iron content, thereby triggering a sharp increase in thelevel of intracellular ROS, and ultimately leading to massive apoptosisof cancer cells. This indicates that the mechanism of GIFT killingcancer cells is the ferroptosis amplified by the gene interferencedesigned by the present invention.

2.5. Antitumor Effect of GIFT Based on Viral Vectors In Vitro(Evaluation of Viral Vectors)

To determine whether the combination of FeNPs with pDMP-Cas13a-U6-gRNAor pDMP-miRNA affects tumor growth in vivo. DMP-Cas13a-U6-gRNA andDMP-miRNA were packaged into AAV vectors to construct recombinantviruses rAAV-DCUg-NT, rAAV-DCUg-Hfl/rAAV-DCUg-mFL, rAAV-DMNeg andrAAV-DMhFL/rAAV-DMmFL. Three cells, KG-1a, WEHI-3 and HL7702, wereinfected with the recombinant viruses, after which the cells wereincubated with or without FeNPs for an additional 72 hours. The resultsshowed that combined treatment of two targeted rAAVs (rAAV-DCUg-hFL/mFLand rAAV-DMhFL/DMmFL) with FeNPs, compared to two non-targeted rAAVs(rAAV-DCUg-NT and rAAV-DMNeg), Caused significant apoptosis of KG-1a andWEHI-3 cells. Whereas rAAV-DCUg-NT and rAAV-DMNeg viruses alone, FeNPs,and the combination of rAAV-DCUg-NT and rAAV-DMNeg viruses with FeNPsdid not significantly affect the growth of these two leukemia cells(FIG. 32 ). In HL7702 cells, treatment of all viruses with FeNPs aloneor in combination did not cause significant apoptosis (FIG. 32 ).

2.6. Iron Nanocarriers (FeNCs)-Based GIFT Inhibits Cancer Cells(Evaluation of Nanosiderophores)

In order to explore whether iron nanoparticles can be used as genecarriers, the gene carrier and iron nanoparticles were combined intoone, as a reagent to achieve GIFT treatment, a PEI-modified Fe₃O₄(FeNCs) was selected as the DNA transfection agent, and the It isdefined as iron nanocarriers (FeNCs). Two GIFT inhibition experiments oncancer cells were performed using two batches of FeNCs (FeNCs-1 andFeNCs-2).

In the first GIFT inhibition experiment based on FeNCs, four plasmids(pDCUg-NT, pDCUg-hFL, pDMNeg and pDMhFL) were loaded with FeNCs-1 toprepare FeNCs loaded with plasmid DNA (FeNCs@DNA) to obtain FeNCs−1@pDCUg-NT, FeNCs-1@pDCUg-hFL, FeNCs-1@pDMNeg and FeNCs-1@pDMhFL. Bloodcancer cells KG-1a were first treated with these FeNCs-1@DNA for DNAtransfection, and then the cells were retreated with 50 μg/mL FeNPs, andcell growth was detected by acridine orange/ethidium bromide staining atdifferent time points. The results showed that FeNPs alone and FeNCs@DNAtreatment of cells did not significantly affect cell growth (FIG. 33 );however, when cells were co-treated with FeNCs-1@pDCUg-hFL andFeNCs-1@pDMhFL with FeNPs, cells Significant time-dependent deathoccurred (FIG. 33 ), while co-treatment of cells with FeNCs-1@pDCUg-NTand FeNCs-1@pDMNeg with FeNPs did not have a significant effect on cellgrowth (FIG. 33 ). Solid tumor cells HepG2 were also treated using thesame method with similar results (FIG. 34 ). It shows that the use ofiron nanoparticles as a gene transfection agent can also introduce geneinterference vectors into cells, resulting in the effect of GIFT oninhibiting cancer cells. In addition, this experiment also showed thatalthough FeNCs-1 is also nano-iron, due to the limited dose used,treating both cancer cells with FeNCs-1@pDCUg-hFL and FeNCs-1@pDMhFLalone did not affect their growth.

Obviously, it is cumbersome to use two kinds of bulk nanoparticles(FeNPs and FeNCs). Therefore, in the second FeNCs-based GIFT inhibitionexperiment on cancer cells, two plasmids (pDMNeg and pDMhFL) were mixedwith FeNCs-1 and FeNCs-2, respectively, to prepare FeNCs@DNA to obtainFeNCs-1@pDMhFL and FeNCs-2@pDMhFL. The leukemia cells KG-1a were treatedwith the prepared FeNCs-1@pDMhFL and FeNCs-2@pDMhFL at a dose of 50μg/mL. The results showed that neither FeNCs nor DNA treatment of cellshad a significant effect on cell growth (FIG. 35 ); however, when cellswere treated with FeNCs-1@pDMhFL and FeNCs-2@pDMhFL, cells experiencedsignificant time-dependent death (FIG. 35 ). In order to furtherinvestigate the stability of FeNCs@DNA, that is, whether DNA would falloff from FeNCs in a short time, affecting the efficiency of transfectedcells in vivo, the prepared FeNCs-1@pDMhFL and FeNCs-2@pDMhFL wereplaced for 24 hours (FeNCs-1@pDMhFL). @DNA has a certain time to reachcancer cells after intravenous injection), and then it is used to treatcells. The results showed that FeNCs@DNA had a similar killing effect oncancer cells after placement (FIG. 35 ).

2.7. In Vivo Antitumor Effect of GIFT

Animal experiments: WEHI-3 cells were transplanted to BALB/c femalemice, and three batches of animal experiments were carried out. Tumorinhibition experiments based on rAAV-DCUg-mFL were carried out in thesecond batch of animal experiments. Six groups of tumor-bearing micewere treated with different treatments, including PBS, FeNPs,rAAV-DCUg-NT, rAAV-DCUg-NT+FeNPs, rAAV-DCUg-mFL, andrAAV-DCUg-mFL+FeNPs. The results showed that the rAAV-DCUg-mFL+FeNPstreatment group produced a significant tumor-inhibiting effect, whilethe other treatment groups did not produce a significanttumor-inhibiting effect (FIG. 36A and FIG. 36B). Tumor inhibitionexperiments based on rAAV-DMmFL were carried out in the second batch ofanimal experiments. Five groups of tumor-bearing mice were treated withdifferent treatments, including FeNP, rAAV-DMNeg, rAAV-DMmFL,rAAV-DMNeg+FeNPs, and rAAV-DMmFL+FeNPs. The results showed that therAAV-DMmFL+FeNPs treatment group had a significant tumor-suppressingeffect, while the other treatment groups had no significanttumor-suppressing effect (FIG. 36A and FIG. 36B).

In the third batch of animal experiments, tumor inhibition experimentsbased on iron nanoparticles directly loaded with plasmid DNA wereinvestigated. In this experiment, FeNCs, a DNA transfection reagentbased on iron oxide nanomaterials, were used for in vivo DNA delivery.Six groups of tumor-bearing mice were treated with different treatments,including PBS, FeNCs, pAAV-DMNeg+FeNCs, pAAV-DMmFL+FeNCs,pAAV-DCUg-NT+FeNCs, and pAAV-DCUg-mFL+FeNCs. The results showed thatFeNCs loaded with two targeting plasmids (pAAV-DCUg-mFL and pAAV-DMmFL)significantly inhibited tumor growth, while FeNCs alone weresignificantly inhibited with two non-targeting plasmids (pAAV-DCUg-NT),and pAAV-DMNeg) FeNCs did not produce significant tumor growthinhibition (FIG. 37A and FIG. 37B).

To further demonstrate the tumor-specific expression of rAAV vectors invivo, the abundance of rAAV DNA (first and second animal experiments)and pAAV DNA (third batch of animal experiments) in various tissues inthree batches of animal experiments was detected, and the expression ofCas13a and target genes. qPCR detection showed that rAAV DNA and pAAVDNA were distributed to different degrees in various tissues, but thehighest distribution level was in tumor tissue, followed by liver (FIG.36C, FIG. 37C). qPCR detection also showed that Cas13a mRNA onlyappeared in tumor tissue, that is, Cas13a was only expressed in tumortissue (FIG. 36D, FIG. 37D); in addition, the two target genes FPN andLcn2 were expressed to different degrees in various tissues. Theexpression of FPN gene was highest in liver and kidney tissues, whilethe expression of Lcn2 gene was highest in tumor tissues (FIGS. 36E and36F, FIG. 37E and FIG. 37F). After various treatments, the expression ofthese two target genes was significantly down-regulated only in tumorsby treatments containing rAAV-DCUg-mFL, rAAV-DMmFL, pAAV-DCUg-mFL, andpAAV-DMmFL (FIGS. 37E and 37F). These results indicate that Cas13a andmiRNA controlled by DMP are activated and expressed only in tumortissues in vivo, thereby knocking down the expression of target genesonly in tumor tissues, reflecting the tumor-specific activation of DMPpromoter in vivo.

2.8. GIFT Targeting Other Genes Inhibits Cancer Cell Growth In Vitro

In order to further investigate whether GIFT targeting other genes alsohas similar anti-tumor effects, and to further the mechanism of GIFTkilling tumor cells, pDMP-miR vectors targeting other five genes weredesigned and constructed, namely FSP1, FTH1, GPX4, NRF2 and SLC7A11, andmiRNAs targeting two targets were designed for each gene. Theconstructed vectors were named pDMhFSP1-1, pDMhFSP1-2, pDMhFTH1-1,pDMhFTH1-2, pDMhGPX4-1, pDMhGPX4-2, pDMhNRF2-1, pDMhNRF2-2,pDMhSLC7A11-1 and pDMhSLC7A11-2, respectively. The selected five genesare closely related to cellular iron metabolism, ROS regulation andferroptosis. Among them, GPX4 and FSP1 are ferroptosis-related genes,FTH1 is a ferritin-encoding gene involved in intracellular iron storage,and NRF2 is a redox-related transcription factor, SLC7A11, is a cystinemembrane import protein involved in the synthesis of the intracellularreducing agent glutathione. FTH1 helps to store excess iron ions incells to maintain intracellular iron homeostasis; SLC7A11 importscystine into cells so that cells can synthesize glutathione to removeintracellular ROS; it is speculated to use pDMP targeting these genesKnockdown of their expression in cancer cells by -miR vector isbeneficial to increase the intracellular iron ion content and increasethe ROS level when FeNPs treat cells, which is beneficial to promoteferroptosis.

By selecting one leukemia cell KG-1a, two solid tumor cells HepG2 (humanhepatoma cells) and BGC823 (human gastric cancer cells), and twocorresponding human normal cells HL7702 (human normal hepatocytes) andGES-1 (human normal gastric mucosal epithelial cells) to test the abovevectors. The cell viability was measured by acridine orange/ethidiumbromide staining and CCK-8 method of cells treated at different timepoints. The results showed that each carrier alone had no significanteffect on the growth of the above five cells (FIG. 38 -FIG. 42 ).), andwhen they were used in combination with FeNPs, they produced significanttime-dependent killing effects on all 3 cancer cells (KG-1a, HepG2,BGC823) (FIG. 38 -FIG. 40 ), but not on both normal cells significanteffect (FIG. 41 , FIG. 42 ). The negative control vector pDMNeg alone orin combination with FeNPs had no significant effect on the growth of theabove five types of cells (FIG. 38 -FIG. 42 ). In addition,co-transfection (miFFGNS) experiments of 5 gene pDM vectors were alsoperformed, and it was found that this co-transfection couldsignificantly inhibit 3 types of cancer cells (KG-1a, HepG2, BGC823)even in the absence of FeNPs. However, when this co-transfection existsin FeNPs, it has a very significant killing effect on cancer cells, andits effect The killing effect of each vector alone in combination withFeNPs on cancer cells was exceeded (FIG. 38 -FIG. 40 ). It shows thatthe five genes have synergistic effect. The viability of various cellsunder various treatments was determined by the CCK8 method, and theresults were consistent with the results of acridine orange/ethidiumbromide staining, and more clearly showed that the five genes hadsignificant synergistic effects (FIG. 43 ).

What is claimed is:
 1. A composition for killing cancer cells,comprising a gene interference vector and iron nanoparticles, whereinthe gene interference vector is a CRISPR/Cas13a expression vector or amicroRNA expression vector controlled by a cancer cell specific promoterDMP; the Cas13a-gRNA expressed by the CRISPR/Cas13a expression vector orthe microRNA expressed by the microRNA expression vector is configuredto inhibit, in a targeted manner, intracellular iron metabolism andexpression of reactive oxygen species-related genes; and the ironnanoparticles are iron nanomaterials that are configured to be degradedafter entering cells to generate iron ions and to increase the level ofintracellular reactive oxygen species levels.
 2. (canceled)
 3. Thecomposition for killing cancer cells according to claim 1, wherein thecancer cell specific promoter DMP is a NF-κB-specific promoter, which iscomposed of NF-κB; the κB decoy and a minimal promoter are connected toform, the promoter is configured to activate its downstream genes to beexpressed in various cancer cells, but not in normal cells; the DMPpromoter can control the expression of CRISPR/Cas13a or microRNAexpression vector in cancer cells specific expression in.
 4. Thecomposition for killing cancer cells according to claim 1, wherein inthe CRISPR/Cas13a expression vector, the expression of Cas13a iscontrolled by the DMP promoter, and the expression of gRNA is controlledby the U6 promoter; the expression of the microRNA is controlled by theDMP promoter in the microRNA expression vector.
 5. The composition forkilling cancer cells according to claim 1, wherein the DNA sequence ofthe functional element of the CRISPR/Cas13a expression vector is shownin SEQ ID NO.1; the DNA sequence of the functional element of themicroRNA expression vector is shown in SEQ ID NO.2.
 6. The compositionfor killing cancer cells according to claim 1, wherein the CRISPR/Cas13aor microRNA expression vector is configured to express either a singlegene-targeting gRNA or microRNA; or as a combined CRISPR/Cas13a ormicroRNA expression vector to express gRNA or microRNA targetingmultiple genes.
 7. The composition for killing cancer cells according toclaim 2, wherein the iron metabolism and reactive oxygen species-relatedgenes is selected from FPN, LCN2, FSP1, FTH1, GPX4, NRF2 and SLC7A11genes.
 8. The composition for killing cancer cells according to claim 7,wherein the CRISPR/Cas13a or microRNA expression vector is configured toexpress gRNA or microRNA targeting a group of genes selected from FPN,LCN2, FSP1, FTH1, GPX4, NRF2 and SLC7A11 genes, in which gRNA can form afirst complex with Cas13a protein, and microRNA can form a secondcomplex with RISC, and both first and second complexes can targetcleavage of the mRNA of the group of genes, causing a reduction ofexpression level of proteins encoded by the group of genes.
 9. Thecomposition for killing cancer cells according to claim 7, whereintarget binding sequences of the gRNAs targeting FPN and LCN2 are: (FPN)5′-CACCG CAAAG TGCCA CATCC GATCT CCC-3′  and (LCN2)5′-TAACT CTTAA TGTTG CCCAG CGTGA ACT-3′;

target binding sequences of the microRNAs targeting FPN, LCN2, FSP1,FTH1, GPX4, NRF2 and SLC7A11 genes are: (FPN)5′-TCTAC CTGCA GCTTA CATGA T-3′, (LCN2) 5′-TAATG TTGCC CAGCG TGAAC T-3′,(FSP1) 5′-CAAAC AAACA AATAA AGTGG A-3′, (FSP1)5′-TAAAC AAACA AACAA ATAAA G-3′, (FTH1) 5′-ATCCC AAGAC CTCAA AGACA A-3′,(FTH1) 5′-TAAGG AATCT GGAAG ATAGC C-3′, (GPX4)5′-TTCAG TAGGC GGCAA AGGCG G-3′, (GPX4) 5′-AGGAA CTGTG GAGAG ACGGT G-3′,(NRF2) 5′-TACTG ATTCA ACATA CTGAC A-3′, (NRF2)5′-TTTAC ACTTA CACAG AAACT A-3′, (SLC7A11)5′-AAATG ATACA GCCTT AACAC A-3′ and (SLC7A11)5′-TTGAG TTGAG GACCA GTTAG T-3′.


10. The composition for killing cancer cells according to claim 1,wherein the iron nanoparticles are FeNPs or FeNCs.
 11. The compositionfor killing cancer cells according to claim 1, wherein under thecombined action of gene interference vector and iron nanoparticles, thelevels of iron ions and reactive oxygen species in cancer cells can besharply increased, inducing significant ferroptosis in cancer cells. 12.The composition for killing cancer cells according to claim 1, whereinthe gene interference vector is configured to be administered in vivo ina form of viral vectors including adeno-associated virus and othernon-viral vectors including nanocarriers; and the iron nanoparticles isconfigured to be administered in vivo either as a separate chemicalmaterial or as a nanocarrier of gene interference vector for in vivoadministration concurrently.
 13. The composition for killing cancercells according to claim 1, wherein the composition for killing cancercells of claim 1 is used in a preparation of novel cancer therapeuticagents.